Methods and compositions for treating disorders

ABSTRACT

Compounds and compositions for treating disorders related to TRPA1 are described herein.

CLAIM OF PRIORITY

This application claims priority from U.S. Ser. No. 60/945,840, filedJun. 22, 2007 and U.S. Ser. No. 60/945,866, filed Jun. 22, 2007, each ofwhich is incorporated herein by reference in its entirety.

BACKGROUND

A variety of ion channel proteins exist to mediate ion flux acrosscellular membranes. The proper expression and function of ion channelproteins is essential for the maintenance of cell function,intracellular communication, and the like. Numerous diseases are theresult of misregulation of membrane potential or aberrant calciumhandling. Given the central importance of ion channels in modulatingmembrane potential and ion flux in cells, identification of agents thatcan promote or inhibit particular ion channels are of great interest asresearch tools and as possible therapeutic agents.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for treating orpreventing conditions such as pain by modulating the activity of theTRPA1 channel. The compounds described herein modulate the function ofTRPA1 by inhibiting a TRPA1-mediated ion flux or by inhibiting theinward current, the outward current, or both currents mediated by TRPA1.The inhibition of a particular current is the ability to inhibit orreduce such current (e.g., inward and/or outward) in an in vitro or anin vivo assay. The following articles are exemplary of the state of theart regarding the structure and function of TRPA1 (Jordt et al. (2004)Nature 427:260-265; Bautista et al., (2005) PNAS: 102(34):12248-12252).The foregoing articles are incorporated by reference in their entirety.

One aspect of the present invention relates to a method for treating orpreventing a condition involving activation of TRPA1 or for whichreduced TRPA1 activity can reduce the severity by administering a TRPA1antagonist that inhibits TRPA1-mediated current and/or TRPA1-mediatedion flux. Described in greater detail below are TRPA1 antagonists thathave measured IC₅₀'s for inhibition of TRPA1 of 10 micromolar or less, 5micromolar or less, 2 micromolar or less, 1 micromolar or less, 500nanomolar or less, 200 nanomolar or less, 100 nanomolar or less, andeven 10 nanomolar or less. In certain embodiments, the TRPA1 antagonistinhibit one or both of inward and outward TRPA1-mediated current with anIC₅₀ of 1 micromolar or less, and more preferably with an IC₅₀ of 500nanomolar or less, 200 nanomolar or less, 100 nanomolar or less, 25nanomolar or less and even 10 nanomolar or less. In certain embodiments,the TRPA1 antagonist inhibits at least 95% of TRPA1-mediated current orTRPA1-mediated ion flux when administered at 5 micromolar or less, andeven more preferably at 1 micromolar or less.

In certain embodiments, the subject TRPA1 antagonists inhibit TRPA1 withan IC₅₀ at least one order of magnitude lower than its IC₅₀ forinhibition of one or more of TRPV5, TRPV6, NaV 1.2, TRPV1, mitochondrialuniporter and hERG channel activities, and even more preferably two oreven three orders of magnitude lower.

In certain embodiments, the subject TRPA1 antagonists are at least 10,20, 30, 40, or 50 fold selective for inhibiting TRPA1 activity over thatof one or more of TRPV5, TRPV6, NaV 1.2, TRPV1, mitochondrial uniporter,or hERG channel activities. In other words, the antagonist inhibitsTRPA1 activity (one or more functions of TRPA1) 10, 20, 30, 40, or 50times more potently than that of one or more of the foregoing channels.

In certain embodiments, the subject TRPA1 antagonists inhibit TRPA1 withan IC₅₀ at least one order of magnitude more potent than its Ki for theAMPA receptor. In certain other embodiments, the subject TRPA1antagonists inhibit TRPA1 with an IC₅₀ at least two orders of magnitude,or even three orders of magnitude, or four orders of magnitude morepotent than its Ki for the AMPA receptor. In certain embodiments, thesubject TRPA1 antagonists do not appreciably bind the AMPA receptor. Inother words, the subject antagonists inhibit TRPA1 with a particularIC₅₀ and, when administered at that concentration, the antagonist doesnot appreciably bind AMPA receptor (e.g., does specifically andappreciably bind the AMPA receptor). In certain embodiments, compoundsof the invention inhibit a TRPA1-mediated current with an IC₅₀ that ismore potent than its Ki for the AMPA receptor. In such embodiments, theability of the subject TRPA1 inhibitors to decrease pain would thus beindependent of binding to and modulation of thealpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptorwhich has been implicated in neuropathic pain reception.

In certain embodiments, the TRPA1 antagonists inhibit TRPA1 with an IC₅₀at least one order of magnitude lower than its IC₅₀ for inhibition ofTRPV1, and even more preferably two or even three orders of magnitudelower. In certain embodiments, the subject TRPA1 antagonists can beselected for selectivity for TRPA1 versus TRPV1 on the basis of havingIC₅₀ for TRPV1 inhibition greater than 10 micromolar.

In certain embodiments, the TRPA1 antagonists inhibit one or more ofTRPV2, TRPV4, TRPV3 and/or TRPM8 with an IC₅₀ of 10 micromolar or less.

In certain embodiments, the TRPA1 antagonist has a therapeutic index(T.I.) for treating the condition with the compound of 10 or greater,and even more preferably has a T.I. of at least 25, 50 or even 100.

In preferred embodiments, the TRPA1 inhibitor has an IC₅₀ for TRPA1inhibition that, at that concentration, does not cause QT intervalelongation in the patient nor alter temperature regulation in thepatient.

In certain embodiments, the TRPA1 inhibitor is used to treat orameliorate pain. Exemplary classes of pain that can be treated using aTRPA1 inhibitor include, but are not limited to nociceptive pain,inflammatory pain, and neuropathic pain. Pain that can be treated with aTRPA1 inhibitor can be chronic or acute.

In certain embodiments, the TRPA1 inhibitor is used to treat orameliorate the symptoms of incontinence.

In certain embodiments, the TRPA1 inhibitor is non-narcotic and haslittle or no narcotic side-effects. In certain other embodiments, theTRPA1 inhibitor can be used to treat or ameliorate pain with fewerside-effects than narcotic pain relievers. Exemplary side-effects thatmay be substantially absent at effective dosages of TRPV3 inhibitorsinclude one or more of exopthalmos, catalepsy, disruption of gutmotility, and inhibition of sensation in non-injured areas of the body.

In certain embodiments, a TRPA1 inhibitor used in the treatment of anyof the diseases or indications disclosed herein has one or more of thestructural or functional characteristics disclosed herein.

In one aspect, the invention features a compound of formula (I),

wherein,

R¹ and R² are each independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl, each of which is optionally substituted with 1-4 R⁵;

L is NR⁶SO₂, SO₂NR⁶, C(O)NR⁶, NR⁶C(O), OC(O)NR⁶, NR⁶C(O)O, NR⁶C(O)NR⁶,S, S(O), S(O)₂, NR⁶, CH₂, O, C(O), C(O)NS(O)₂, S(O)₂NC(O), heteroaryl,or cyclyl;

R³ is cyclyl, heterocyclyl, aryl, heteroaryl, each of which isoptionally substituted with 1-4R⁷;

each R⁵ is independently halo, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, cyano, nitro, amido, alkylamido, dialkylamido, thioyl,sulfonyl, cyclyl, heterocyclyl, aryl, or heteroaryl;

each R⁶ is independently H, C₁-C₆ alkyl, C₁-C₆ alkenyl, hydroxyC₁-C₆alkyl, alkoxyC₁-C₆ alkyl, cyanoalkyl, haloalkyl, arylalkyl, S(O)alkyl,acyl, amino, amidyl, or S(O)₂H, aryl, alkoxyaryl;

each R⁷ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,cyclyl, heterocyclyl, aryl, heteroaryl, cyclylalkyl, heterocyclylalkyl,arylalkyl, heteroarylalkyl, halo, hydroxyl, alkoxy, aryloxy, arylalkoxy,amino, alkylamino, dialkylamino, thioyl, alkylthioyl, sulfonyl,sulfonamidyl (e.g., where the nitrogen of the sulfonamide is substitutedby an alkyl, or where the nitrogen of the sulfonamide together with twocarbons to which it is attached, forms a ring), amido (e.g., where thenitrogen of the amide is substituted by an alkyl, or where the nitrogenof the amide together with two carbons to which it is attached, forms aring), urea, sulfonylurea, acyl, —C(O)aryl, —NHC(O)aryl, —C(O)NHaryl,—C(O)OH, —C(O)Oalkyl, nitro, cyano, each of which is optionallysubstituted with 1-3 R⁸;

each R⁸ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, C₁-C₆ haloalkyl, hydroxyl, alkoxy, aryloxy, amino, alkylamino,dialkylamino, thioyl, sulfonyl, sulfonamidyl, amido(e.g., where thenitrogen of the amide is substituted by an alkyl, or where the nitrogenof the amide together with two carbons to which it is attached, forms aring), C(O)OH, —C(O)Oalkyl, urea, sulfonylurea acyl, nitro, cyano,cyclyl, heterocyclyl, aryl, or heteroaryl; optionally substituted with1-3 C₁-C₆ alkyl, C₁-C₆ haloalkyl, or halo;

R⁹ is independently H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, C₁-C₆ haloalkyl, hydroxyl, alkoxy, aryloxy, arylalkoxy, amino,alkylamino, dialkylamino, thioyl, alkylthioyl, sulfonyl, sulfonamidyl,amido, urea, sulfonylurea, acyl, nitro, cyano, each of which isoptionally substituted with 1-3 R⁸;

each m and n are independently 0, 1, 2, 3, 4, 5, or 6.

In some embodiments, when L is heteroaryl or cyclopropyl, n is at least1.

In some embodiments, R¹ is C₁-C₆ alkyl, for example, methyl.

In some embodiments, R¹ is further substituted by a dialkyl amine, forexample, a dimethyl amine.

In some embodiments, wherein R¹ is

In some embodiments, R¹ is C₁-C₆ alkyl substituted by heterocyclyl, forexample a nitrogen containing heterocyclyl such as morpholinyl.

In some embodiments, R² is C₁-C₆ alkyl, for example, methyl.

In some embodiments, R² is further substituted by a dialkyl amine, forexample, a dimethyl amine.

In some embodiments, wherein R² is

In some embodiments, R² is C₁-C₆ alkyl substituted by heterocyclyl, forexample, a nitrogen containing heterocyclyl such as morpholinyl.

In some embodiments, both R¹ and R² are C₁-C₆ alkyl, for example, bothR¹ and R² are methyl.

In some embodiments, R³ is monocyclic, for example a monocyclic cyclyl,a monocyclic aryl, a monocyclic heterocyclyl, or a monocyclicheteroaryl.

In some embodiments, R³ is aryl, for example, phenyl.

In some embodiments, R³ is phenyl substituted by 1-3 R⁷. In someembodiments, R⁷ is Me, OMe, or halo. In some embodiments, at least 1 R⁷is positioned in the para position.

In some embodiments, R³ is phenyl substituted by 1 R⁷. In someembodiments, R⁷ is Me, OMe, or halo. In some embodiments, R⁷ ispositioned in the para position, for example, when R⁷ is Me, OMe, orhalo, e.g., R⁷ is methyl.

In some embodiments, R³ is

In some embodiments, R³ is heterocyclyl, for example, a nitrogencontaining heterocyclyl and/or a 5 membered heterocyclyl. In someembodiments, R³ is substituted by 1-3 R⁷. In some embodiments, at least1 R⁷ is in the 3 position of the 5 membered ring. In some embodiments,R⁷ is Me, OMe, or halo.

In some embodiments, R³ is substituted by 1 R⁷, for example, Me, OMe, orhalo. In some embodiments, R⁷ is in the 3 position of the 5 memberedring, for example, when R⁷ is Me, OMe, or halo.

In some embodiments, R³ is a 6 membered heterocyclyl, for example, R³ is

In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, R³ is heteroaryl, for example, a 5 or 6 memberedheteroaryl, e.g., a 5 membered heteroaryl. In some embodiments, R³ issubstituted by 1-3 R⁷, for example, Me, OMe, or halo. In someembodiments, at least 1 R⁷ is in the 3 position of the 5 membered ring,for example when R⁷ is Me, OMe, or halo. In some embodiments, R³ issubstituted by 1 R⁷, for example, when R⁷ is Me, OMe, or halo. In someembodiments, R⁷ is in the 3 position of the 5 membered ring, forexample, when R⁷ is Me, OMe, or halo. In some embodiments, R⁷ is in the4 position of the 5 membered ring, for example, when R⁷ is Me, OMe, orhalo.

In some embodiments, R³ is a nitrogen containing heteroaryl, forexample,

In some embodiments, the 5 membered heteroaryl is substituted by atleast 1 R⁷ (e.g., one or two), for example, is in the 3 or 4 position ofthe 5 membered ring.

In some embodiments, R³ is a 6 membered heteroaryl, for example,substituted by 1-3 R⁷ such as Me, OMe, or halo. In some embodiments, atleast 1 R⁷ is positioned in the para position, for example, when R⁷ isMe, OMe, or halo. In some embodiments, R³ is substituted by 1 R⁷, forexample, when R⁷ is Me, OMe, or halo. In some embodiments, R⁷ ispositioned in the para position, for example, when R⁷ is Me, OMe, orhalo.

In some embodiments, R³ is a 6 membered, nitrogen containing heteroaryl.

In some embodiments, R³ is

In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, R³ is a 6 membered heteroaryl containing 2nitrogens, e.g.,

In some embodiments, R³ is substituted by 1-3 R⁷.

In some embodiments, R³ is a heteroaryl or heterocycyl having two fusedrings. In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, R³ is a heteroaryl or heterocycyl having threefused rings. In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, each R⁷ is independently C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, halo, hydroxyl, alkoxy, aryloxy, arylalkoxy,amino, alkylamino, dialkylamino, thioyl, alkylthioyl, sulfonyl,sulfonamidyl, amido (e.g., where the nitrogen of the amide issubstituted by an alkyl, or where the nitrogen of the amide togetherwith two carbons to which it is attached, forms a ring), urea,sulfonylurea, acyl, nitro, cyano, each of which is optionallysubstituted with 1-3 R⁸;

In some embodiments, L is L is NR⁶SO₂, SO₂NR⁶, C(O)NR⁶, NR⁶C(O),OC(O)NR⁶, NR⁶C(O)O, NR⁶C(O)NR⁶, S, S(O), S(O)₂, C(O)NS(O)₂, S(O)₂NC(O),heteroaryl, or cyclyl.

In some embodiments, L is L is NR⁶SO₂, SO₂NR₆, OC(O)NR₆, NR₆C(O)O,NR6C(O)NR6, S, S(O), S(O)2, C(O)NS(O)2, S(O)2NC(O), heteroaryl, orcyclyl.

In some embodiments, L is NR⁶SO₂ or SO₂NR⁶, OC(O)NR⁶, NR⁶C(O)O,NR⁶C(O)NR₆. In some embodiments, R⁶ is H.

In some embodiments, L is OC(O)NR⁶ or NR⁶C(O)O. In some embodiments, R⁶is H.

In some embodiments, L is NR⁶C(O)NR⁶. In some embodiments, R⁶ is H.

In some embodiments, L is cyclyl or heterocyclyl, for example,cyclopropyl.

In some embodiments, L is C(O).

In some embodiments, R⁹ is H.

In some embodiments, R⁹ is halo, for example, chloro.

In some embodiments, m is 1.

In some embodiments, n is 2.

In some embodiments, m is 1 and n is 2.

In some embodiments, n is 0.

In some embodiments, m is 1 and n is 0.

In some embodiments, m+n≦6.

In some embodiments, the compound has one of the formula below:

In one aspect, the compound is a compound of formula (Ia)

wherein,

R¹ and R² are each independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl, each of which is optionally substituted with 1-4 R⁵;

L is NR₆C(O) or C(O)NR₆;

R³ is cyclyl, heterocyclyl, aryl, heteroaryl, each of which isoptionally substituted with 1-4 R⁷;

each R⁵ is independently halo, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, cyano, nitro, amido(e.g., where the nitrogen of the amideis substituted by an alkyl, or where the nitrogen of the amide togetherwith two carbons to which it is attached, forms a ring), alkylamido,dialkylamido, thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, orheteroaryl;

each R⁶ is independently H, C₁-C₆ alkyl, arylalkyl, S(O)alkyl, acetyl,or S(O)H;

each R⁷ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,cyclyl, heterocyclyl, aryl, heteroaryl, cyclylalkyl, heterocyclylalkyl,arylalkyl, heteroarylalkyl, halo, hydroxyl, alkoxy, aryloxy, arylalkoxy,amino, alkylamino, dialkylamino, thioyl, alkylthioyl, sulfonyl,sulfonamidyl, amido (e.g., where the nitrogen of the amide issubstituted by an alkyl, or where the nitrogen of the amide togetherwith two carbons to which it is attached, forms a ring), urea,sulfonylurea, acyl, —C(O)aryl, —NHC(O)aryl, —C(O)NHaryl, nitro, cyano,each of which is optionally substituted with 1-3 R⁸;

each R⁸ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, C₁-C₆ haloalkyl, hydroxyl, alkoxy, aryloxy, amino, alkylamino,dialkylamino, thioyl, sulfonyl, sulfonamidyl, amido(e.g., where thenitrogen of the amide is substituted by an alkyl, or where the nitrogenof the amide together with two carbons to which it is attached, forms aring), urea, sulfonylurea acyl, nitro, cyano, cyclyl, heterocyclyl,aryl, or heteroaryl; optionally substituted with 1-3 C₁-C₆ alkyl, C₁-C₆haloalkyl, or halo;

each R⁹ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, C₁-C₆ haloalkyl, hydroxyl, alkoxy, aryloxy, arylalkoxy, amino,alkylamino, dialkylamino, thioyl, alkylthioyl, sulfonyl, sulfonamidyl,amido, urea, sulfonylurea, acyl, nitro, cyano, each of which isoptionally substituted with 1-3 R⁸;

-   m is 0, 1, 2, 3, 4, 5, or 6; and-   n is 2, 3, 4, 5, or 6.

In some embodiments, when m is 1, n is 2, L is C(O)NH, and R¹ and R² areboth methyl, R³ is not phenyl. In some embodiments, when L is NR₆C(O), mis at least 2.

In some embodiments, R¹ is C₁-C₆ alkyl, for example, methyl.

In some embodiments, R¹ is further substituted by a dialkyl amine, forexample, a dimethyl amine.

In some embodiments, wherein R¹ is

In some embodiments, R¹ is C₁-C₆ alkyl substituted by heterocyclyl, forexample a nitrogen containing heterocyclyl such as morpholinyl.

In some embodiments, R² is C₁-C₆ alkyl, for example, methyl.

In some embodiments, R² is further substituted by a dialkyl amine, forexample, a dimethyl amine.

In some embodiments, wherein R² is

In some embodiments, R² is C₁-C₆ alkyl substituted by heterocyclyl, forexample, a nitrogen containing heterocyclyl such as morpholinyl.

In some embodiments, both R¹ and R² are C₁-C₆ alkyl, for example, bothR¹ and R² are methyl.

In some embodiments, R³ is monocyclic, for example a monocyclic cyclyl,a monocyclic aryl, a monocyclic heterocyclyl, or a monocyclicheteroaryl.

In some embodiments, R³ is aryl, for example, phenyl.

In some embodiments, R³ is phenyl substituted by 1-3 R⁷. In someembodiments, R⁷ is Me, OMe, or halo. In some embodiments, at least 1 R⁷is positioned in the para position.

In some embodiments, R³ is phenyl substituted by 1 R⁷. In someembodiments, R⁷ is Me, OMe, or halo. In some embodiments, R⁷ ispositioned in the para position, for example, when R⁷ is Me, OMe, orhalo, e.g., R⁷ is methyl.

In some embodiments, R³ is

In some embodiments, R³ is heterocyclyl, for example, a nitrogencontaining heterocyclyl and/or a 5 membered heterocyclyl. In someembodiments, R³ is substituted by 1-3 R⁷. In some embodiments, at least1 R⁷ is in the 3 position of the 5 membered ring. In some embodiments,R⁷ is Me, OMe, or halo.

In some embodiments, R³ is substituted by 1 R⁷, for example, Me, OMe, orhalo. In some embodiments, R⁷ is in the 3 position of the 5 memberedring, for example, when R⁷ is Me, OMe, or halo.

In some embodiments, R³ is a 6 member heterocyclyl, for example, R³ is

In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, R³ is heteroaryl, for example, a 5 or 6 memberedheteroaryl, e.g., a 5 membered heteroaryl. In some embodiments, R³ issubstituted by 1-3 R⁷, for example, Me, OMe, or halo. In someembodiments, at least 1 R⁷ is in the 3 position of the 5 membered ring,for example when R⁷ is Me, OMe, or halo. In some embodiments, R³ issubstituted by 1 R⁷, for example, when R⁷ is Me, OMe, or halo. In someembodiments, R⁷ is in the 3 position of the 5 membered ring, forexample, when R⁷ is Me, OMe, or halo. In some embodiments, R⁷ is in the4 position of the 5 membered ring, for example, when R⁷ is Me, OMe, orhalo.

In some embodiments, R³ is a nitrogen containing heteroaryl, forexample,

In some embodiments, the 5 membered heteroaryl is substituted by atleast 1 R⁷ (e.g., one or two), for example, in the 3 or 4 position ofthe 5 membered ring.

In some embodiments, R³ is a 6 membered heteroaryl, for example,substituted by 1-3 R⁷ such as Me, OMe, or halo. In some embodiments, atleast 1 R⁷ is positioned in the para position, for example, when R⁷ isMe, OMe, or halo. In some embodiments, R³ is substituted by 1 R⁷, forexample, when R⁷ is Me, OMe, or halo. In some embodiments, R⁷ ispositioned in the para position, for example, when R⁷ is Me, OMe, orhalo.

In some embodiments, R³ is

In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, R³ is a 6 membered heteroaryl containing 2nitrogens, e.g.,

In some embodiments, R³ is substituted by 1-3 R⁷.

In some embodiments, R³ is a heteroaryl or heterocycyl having two fusedrings. In some embodiments, R³ is substituted by 1-4 R⁷. In someembodiments, R³ is a heteroaryl or heterocycyl having three fused rings.In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, each R⁷ is independently C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, halo, hydroxyl, alkoxy, aryloxy, arylalkoxy,amino, alkylamino, dialkylamino, thioyl, alkylthioyl, sulfonyl,sulfonamidyl, amido (e.g., where the nitrogen of the amide issubstituted by an alkyl, or where the nitrogen of the amide togetherwith two carbons to which it is attached, forms a ring), urea,sulfonylurea, acyl, nitro, cyano, each of which is independentlysubstituted with 1-3 R⁸.

In some embodiments, L is C(O)NR⁶.

In some embodiments, L is NR⁶C(O).

In some embodiments, R⁹ is H.

In some embodiments, R⁹ is halo, for example, chloro.

In some embodiments, m is 1.

In some embodiments, n is 2.

In some embodiments, m is 1 and n is 2.

In some embodiments, n is 0.

In some embodiments, m is 1 and n is 0.

In some embodiments, m+n≦6.

In some preferred embodiments, the compound is a compound of formula(Ia′)

In one aspect, the invention features a compound of formula (Ib)

wherein,

R¹ and R² are each independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl, each of which is optionally substituted with 1-4 R⁵;

L is NR₆C(O) or C(O)NR₆;

R³ is cyclyl, heterocyclyl, aryl, heteroaryl, each of which isoptionally substituted with 1-4 R⁷;

each R⁵ is independently halo, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, cyano, nitro, amido (e.g., where the nitrogen of the amideis substituted by an alkyl, or where the nitrogen of the amide togetherwith two carbons to which it is attached, forms a ring), alkylamido,dialkylamido, thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, orheteroaryl;

each R⁶ is independently H, C₁-C₆ alkyl, arylalkyl, S(O)alkyl, acetyl,or S(O)H;

each R⁷ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, cyclylalkyl, heterocyclylalkyl, arylalkyl, heteroarylalkyl,hydroxyl, alkoxy, aryloxy, arylalkoxy, amino, alkylamino, dialkylamino,thioyl, alkylthioyl, sulfonyl, sulfonamidyl, amido (e.g., where thenitrogen of the amide is substituted by an alkyl, or where the nitrogenof the amide together with two carbons to which it is attached, forms aring), urea, sulfonylurea, acyl, —C(O)aryl, —NHC(O)aryl, —C(O)NHaryl,nitro, cyano, each of which is optionally substituted with 1-3 R⁸;

each R⁸ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, C₁-C₆ haloalkyl, hydroxyl, alkoxy, aryloxy, amino, alkylamino,dialkylamino, thioyl, sulfonyl, sulfonamidyl, amido (e.g., where thenitrogen of the amide is substituted by an alkyl, or where the nitrogenof the amide together with two carbons to which it is attached, forms aring), urea, sulfonylurea, acyl, nitro, cyano, cyclyl, heterocyclyl,aryl, or heteroaryl; optionally substituted with 1-3 C₁-C₆ alkyl, C₁-C₆haloalkyl, or halo;

each R⁹ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, C₁-C₆ haloalkyl, hydroxyl, alkoxy, aryloxy, arylalkoxy, amino,alkylamino, dialkylamino, thioyl, alkylthioyl, sulfonyl, sulfonamidyl,amido, urea, sulfonylurea, acyl, nitro, cyano, each of which isoptionally substituted with 1-3 R⁸;

m is 0, 1, 2, 3, 4, 5, or 6.

In some embodiments, when L is NR₆C(O), m is at least 2.

In some embodiments, R¹ is C₁-C₆ alkyl, for example, methyl.

In some embodiments, R¹ is further substituted by a dialkyl amine, forexample, a dimethyl amine.

In some embodiments, wherein R¹ is

In some embodiments, R¹ is C₁-C₆ alkyl substituted by heterocyclyl, forexample a nitrogen containing heterocyclyl such as morpholinyl.

In some embodiments, R² is C₁-C₆ alkyl, for example, methyl.

In some embodiments, R² is further substituted by a dialkyl amine, forexample, a dimethyl amine.

In some embodiments, wherein R² is

In some embodiments, R² is C₁-C₆ alkyl substituted by heterocyclyl, forexample, a nitrogen containing heterocyclyl such as morpholinyl.

In some embodiments, both R¹ and R² are C₁-C₆ alkyl, for example, bothR¹ and R² are methyl.

In some embodiments, R³ is monocyclic, for example a monocyclic cyclyl,a monocyclic aryl, a monocyclic heterocyclyl, or a monocyclicheteroaryl.

In some embodiments, R³ is aryl, for example, phenyl.

In some embodiments, R³ is phenyl substituted by 1-3 R⁷. In someembodiments, R⁷ is Me, OMe, or halo. In some embodiments, at least 1 R⁷is positioned in the para position.

In some embodiments, R³ is phenyl substituted by 1 R⁷. In someembodiments, R⁷ is Me, OMe, or halo. In some embodiments, R⁷ ispositioned in the para position, for example, when R⁷ is Me, OMe, orhalo, e.g., R⁷ is methyl.

In some embodiments, R³ is

In some embodiments, R³ is heterocyclyl, for example, a nitrogencontaining heterocyclyl and/or a 5 membered heterocyclyl. In someembodiments, R³ is substituted by 1-3 R⁷. In some embodiments, at least1 R⁷ is in the 3 position of the 5 membered ring. In some embodiments,R⁷ is Me, OMe, or halo.

In some embodiments, R³ is substituted by 1 R⁷, for example, Me, OMe, orhalo. In some embodiments, R⁷ is in the 3 position of the 5 memberedring, for example, when R⁷ is Me, OMe, or halo.

In some embodiments, R³ is a 6 membered heterocyclyl, for example, R³ is

In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, R³ is heteroaryl, for example, a 5 or 6 memberedheteroaryl, e.g., a 5 membered heteroaryl. In some embodiments, R³ issubstituted by 1-3 R⁷, for example, Me, OMe, or halo. In someembodiments, at least 1 R⁷ is in the 3 position of the 5 membered ring,for example when R⁷ is Me, OMe, or halo. In some embodiments, R³ issubstituted by 1 R⁷, for example, when R⁷ is Me, OMe, or halo. In someembodiments, R⁷ is in the 3 position of the 5 membered ring, forexample, when R⁷ is Me, OMe, or halo. In some embodiments, R⁷ is in the4 position of the 5 membered ring, for example, when R⁷ is Me, OMe, orhalo.

In some embodiments, R³ is a nitrogen containing heteroaryl, forexample,

In some embodiments, the 5 membered heteroaryl is substituted by atleast 1 R⁷ (e.g., one or two), for example, in the 3 or 4 position ofthe 5 membered ring.

In some embodiments, R³ is a 6 membered heteroaryl, for example,substituted by 1-3 R⁷ such as Me, OMe, or halo. In some embodiments, atleast 1 R⁷ is positioned in the para position, for example, when R⁷ isMe, OMe, or halo. In some embodiments, R³ is substituted by 1 R⁷, forexample, when R⁷ is Me, OMe, or halo. In some embodiments, R⁷ ispositioned in the para position, for example, when R⁷ is Me, OMe, orhalo.

In some embodiments, R³ is

In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, R³ is a 6 membered heteroaryl containing 2nitrogens, e.g.,

In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, R³ is a heteroaryl or heterocycyl having two fusedrings. In some embodiments, R³ is substituted by 1-4 R⁷. In someembodiments, R³ is a heteroaryl or heterocycyl having three fused rings.In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, each R⁷ is independently C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, halo, hydroxyl, alkoxy, aryloxy, arylalkoxy,amino, alkylamino, dialkylamino, thioyl, alkylthioyl, sulfonyl,sulfonamidyl, amido (e.g., where the nitrogen of the amide issubstituted by an alkyl, or where the nitrogen of the amide togetherwith two carbons to which it is attached, forms a ring), urea,sulfonylurea, acyl, nitro, cyano, each of which is optionallysubstituted with 1-3 R⁸.

In some embodiments, L is C(O)NR⁶.

In some embodiments, L is NR⁶C(O).

In some embodiments, R⁹ is H.

In some embodiments, R⁹ is halo, for example, chloro.

In some embodiments, m is 1.

In one aspect, the invention features a compound of formula (Ic)

wherein,

R¹ and R² are each independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl, each of which is optionally substituted with 1-4 R⁵;

L is NR⁶, CH₂, or O;

R³ is cyclyl, heterocyclyl, aryl, heteroaryl, each of which isoptionally substituted with 1-4 R⁷;

each R⁵ is independently halo, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, cyano, nitro, amido (e.g., where the nitrogen of the amideis substituted by an alkyl, or where the nitrogen of the amide togetherwith two carbons to which it is attached, forms a ring), alkylamido,dialkylamido, thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, orheteroaryl;

each R⁶ is independently H, C₁-C₆ alkyl, arylalkyl, S(O)alkyl, acetyl,or S(O)H;

each R⁷ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, hydroxyl, alkoxy, aryloxy, arylalkoxy, cyclylalkyl,heterocyclylalkyl, arylalkyl, heteroarylalkyl, amino, alkylamino,dialkylamino, thioyl, alkylthioyl, sulfonyl, sulfonamidyl, amido (e.g.,where the nitrogen of the amide is substituted by an alkyl, or where thenitrogen of the amide together with two carbons to which it is attached,forms a ring), urea, sulfonylurea, acyl, nitro, cyano, each of which isoptionally substituted with 1-3 R⁸;

each R⁸ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, C₁-C₆ haloalkyl, hydroxyl, alkoxy, aryloxy, amino, alkylamino,dialkylamino, thioyl, sulfonyl, sulfonamidyl, amido (e.g., where thenitrogen of the amide is substituted by an alkyl, or where the nitrogenof the amide together with two carbons to which it is attached, forms aring), urea, sulfonylurea acyl, —C(O)aryl, —NHC(O)aryl, —C(O)NHaryl,nitro, cyano, cyclyl, heterocyclyl, aryl, or heteroaryl; optionallysubstituted with 1-3 C₁-C₆ alkyl, C₁-C₆ haloalkyl, or halo; each R⁹ isindependently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, halo, C₁-C₆haloalkyl, hydroxyl, alkoxy, aryloxy, arylalkoxy, amino, alkylamino,dialkylamino, thioyl, alkylthioyl, sulfonyl, sulfonamidyl, amido, urea,sulfonylurea, acyl, nitro, cyano, each of which is optionallysubstituted with 1-3 R⁸;

m is 1, 2, 3, 4, 5, or 6; and

n is 1, 2, 3, 4, 5, or 6.

In some embodiments, when L is CH₂ and R³ is phenyl, m and n together donot equal 2, 3, or 4. In some embodiments, when L is NR⁶, R³ is notunsubstituted phenyl or phenyl substituted with OMe or C₁-C₆ alkylfurther substituted with C(O)Ar. In some embodiments when L is NR₆ or O,m is at least 2.

In some embodiments, R¹ is C₁-C₆ alkyl, for example, methyl.

In some embodiments, R¹ is further substituted by a dialkyl amine, forexample, a dimethyl amine.

In some embodiments, wherein R¹ is

In some embodiments, R¹ is C₁-C₆ alkyl substituted by heterocyclyl, forexample a nitrogen containing heterocyclyl such as morpholinyl.

In some embodiments, R² is C₁-C₆ alkyl, for example, methyl.

In some embodiments, R² is further substituted by a dialkyl amine, forexample, a dimethyl amine.

In some embodiments, wherein R² is

In some embodiments, R² is C₁-C₆ alkyl substituted by heterocyclyl, forexample, a nitrogen containing heterocyclyl such as morpholinyl.

In some embodiments, both R¹ and R² are C₁-C₆ alkyl, for example, bothR¹ and R² are methyl.

In some embodiments, R³ is monocyclic, for example a monocyclic cyclyl,a monocyclic aryl, a monocyclic heterocyclyl, or a monocyclicheteroaryl.

In some embodiments, R³ is aryl, for example, phenyl.

In some embodiments, R³ is phenyl substituted by 1-3 R⁷. In someembodiments, R⁷ is Me, OMe, or halo. In some embodiments, at least 1 R⁷is positioned in the para position.

In some embodiments, R³ is phenyl substituted by 1 R⁷. In someembodiments, R⁷ is Me, OMe, or halo. In some embodiments, R⁷ ispositioned in the para position, for example, when R⁷ is Me, OMe, orhalo, e.g., R⁷ is methyl.

In some embodiments, R³ is

In some embodiments, R³ is heterocyclyl, for example, a nitrogencontaining heterocyclyl and/or a 5 membered heterocyclyl. In someembodiments, R³ is substituted by 1-3 R⁷. In some embodiments, at least1 R⁷ is in the 3 position of the 5 membered ring. In some embodiments,R⁷ is Me, OMe, or halo.

In some embodiments, R³ is substituted by 1 R⁷, for example, Me, OMe, orhalo. In some embodiments, R⁷ is in the 3 position of the 5 memberedring, for example, when R⁷ is Me, OMe, or halo.

In some embodiments, R³ is a 6 membered heterocyclyl, for example, R³ is

In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, R³ is heteroaryl, for example, a 5 or 6 memberedheteroaryl, e.g., a 5 membered heteroaryl. In some embodiments, R³ issubstituted by 1-3 R⁷, for example, Me, OMe, or halo. In someembodiments, at least 1 R⁷ is in the 3 position of the 5 membered ring,for example when R⁷ is Me, OMe, or halo. In some embodiments, R³ issubstituted by 1 R⁷, for example, when R⁷ is Me, OMe, or halo. In someembodiments, R⁷ is in the 3 position of the 5 membered ring, forexample, when R⁷ is Me, OMe, or halo. In some embodiments, R⁷ is in the4 position of the 5 membered ring, for example, when R⁷ is Me, OMe, orhalo.

In some embodiments, R³ is a nitrogen containing heteroaryl, forexample,

In some embodiments, the 5 membered heteroaryl is substituted by atleast 1 R⁷ (e.g., one or two), for example, in the 3 or 4 position ofthe 5 membered ring.

In some embodiments, R³ is a 6 membered heteroaryl, for example,substituted by 1-3 R⁷ such as Me, OMe, or halo. In some embodiments, atleast 1 R⁷ is positioned in the para position, for example, when R⁷ isMe, OMe, or halo. In some embodiments, R³ is substituted by 1 R⁷, forexample, when R⁷ is Me, OMe, or halo. In some embodiments, R⁷ ispositioned in the para position, for example, when R⁷ is Me, OMe, orhalo.

In some embodiments, R³ is

In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, R³ is a 6 membered heteroaryl containing 2nitrogens, e.g.,

In some embodiments, R³ is substituted by 1-3 R⁷.

In some embodiments, R³ is a heteroaryl or heterocycyl having two fusedrings. In some embodiments, R³ is substituted by 1-4 R⁷. In someembodiments, R³ is a heteroaryl or heterocycyl having three fused rings.In some embodiments, R³ is substituted by 1-4 R⁷.

In some embodiments, each R⁷ is independently C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, halo, hydroxyl, alkoxy, aryloxy, arylalkoxy,amino, alkylamino, dialkylamino, thioyl, alkylthioyl, sulfonyl,sulfonamidyl, amido(e.g., where the nitrogen of the amide is substitutedby an alkyl, or where the nitrogen of the amide together with twocarbons to which it is attached, forms a ring), urea, sulfonylurea,acyl, nitro, cyano, each of which is optionally substituted with 1-3 R⁸;

In some embodiments, L is NR⁶.

In some embodiments, L is O.

In some embodiments, L is CH₂.

In some embodiments, R⁹ is H.

In some embodiments, R⁹ is halo, for example, chloro.

In some embodiments, m is 1.

In some embodiments, n is 2.

In some embodiments, m is 1 and n is 2.

In some embodiments, n is 0.

In some embodiments, m is 1 and n is 0.

In one aspect, the invention features a compound of formula (Id)

wherein

R³ is a 3 membered ring fused heteroaryl, optionally substituted with1-4 R⁷;

each R⁷ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,cyclyl, heterocyclyl, aryl, heteroaryl, cyclylalkyl, heterocyclylalkyl,arylalkyl, heteroarylalkyl, halo, hydroxyl, alkoxy, aryloxy, arylalkoxy,amino, alkylamino, dialkylamino, thioyl, alkylthioyl, sulfonyl,sulfonamidyl, amido (e.g., where the nitrogen of the amide issubstituted by an alkyl, or where the nitrogen of the amide togetherwith two carbons to which it is attached, forms a ring), urea,sulfonylurea, acyl, nitro, cyano, each of which is optionallysubstituted with 1-3 R⁸;

each R⁸ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, C₁-C₆ haloalkyl, hydroxyl, alkoxy, aryloxy, amino, alkylamino,dialkylamino, thioyl, sulfonyl, sulfonamidyl, amido (e.g., where thenitrogen of the amide is substituted by an alkyl, or where the nitrogenof the amide together with two carbons to which it is attached, forms aring), urea, sulfonylurea acyl, —C(O)aryl, —NHC(O)aryl, —C(O)NHaryl,nitro, cyano, cyclyl, heterocyclyl, aryl, or heteroaryl; optionallysubstituted with 1-3 C₁-C₆ alkyl, C₁-C₆ haloalkyl, or halo;

R⁹ is independently H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, C₁-C₆ haloalkyl, hydroxyl, alkoxy, aryloxy, arylalkoxy, amino,alkylamino, dialkylamino, thioyl, alkylthioyl, sulfonyl, sulfonamidyl,amido, urea, sulfonylurea, acyl, nitro, cyano, each of which isoptionally substituted with 1-3 R⁸.

In some embodiments, R³ is substituted with 0, 1 or 3 R⁷, each of whichis independently C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, halo,hydroxyl, alkoxy, acyl, nitro, or cyano.

In one aspect, the invention features a compound of formula (VIII),

wherein,

R¹ and R² are each independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl, each of which is optionally substituted with 1-4 R⁵;

L is NR⁶SO₂, SO₂NR⁶, OC(O)NR⁶, NR⁶C(O)O, NR⁶C(O)NR⁶, NR⁶C(O), C(O)NR⁶,O, C(O), S, S(O), S(O)₂, NR₆, or CH₂,

each of R^(3a) and R^(3b) is independently cyclyl, heterocyclyl, aryl,heteroaryl, each of which is optionally substituted with 1-4 R⁷;

each R⁵ is independently halo, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, cyano, nitro, amido(e.g., where the nitrogen of the amideis substituted by an alkyl, or where the nitrogen of the amide togetherwith two carbons to which it is attached, forms a ring), alkylamido,dialkylamido, thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, orheteroaryl;

each R⁶ is independently H, C₁-C₆ alkyl, C₁-C₆ alkenyl, hydroxyC₁-C₆alkyl, alkoxyC₁-C₆ alkyl, cyanoalkyl, haloalkyl, arylalkyl, S(O)alkyl,acyl, amino, amidyl, or S(O)₂H, aryl, alkoxyaryl;

each R⁷ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, hydroxyl, alkoxy, oxo, aryl, heteroaryl, cyclyl, heterocyclyl,arylalkyl, heteroarylalkyl, cyclylalkyl, heterocyclylalkyl, aryloxy,arylalkoxy, amino, alkylamino, dialkylamino, thioyl, alkylthioyl,sulfonyl, sulfonamidyl, amido (e.g., where the nitrogen of the amide issubstituted by an alkyl, or where the nitrogen of the amide togetherwith two carbons to which it is attached, forms a ring), hydroxylalkoxyl, alkoxy —C(O)OH, —C(O)Oalkyl, urea, sulfonylurea, acyl, nitro,cyano, each of which is optionally substituted with 1-3 R⁸;

each R⁸ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,aryl, heteroaryl, cyclyl, halo, hydroxyl, alkoxy, oxo, aryloxy, amino,alkylamino, dialkylamino, C(O)OH, —C(O)Oalkyl, thioyl, sulfonyl,sulfonamidyl, amido (e.g., where the nitrogen of the amide issubstituted by an alkyl, or where the nitrogen of the amide togetherwith two carbons to which it is attached, forms a ring), urea,sulfonylurea, acyl, nitro, cyano, cyclyl, heterocyclyl, aryl, orheteroaryl;

R⁹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, halo, C₁-C₆haloalkyl, hydroxyl, alkoxy, aryloxy, arylalkoxy, amino, alkylamino,dialkylamino, thioyl, alkylthioyl, sulfonyl, sulfonamidyl, amido, urea,sulfonylurea, acyl, nitro, cyano, each of which is optionallysubstituted with 1-3 R⁸;

m is 1, 2, 3, 4, 5, or 6.

In some embodiments, m is at least 2 when L is connected to themethylene carbon via a heteroatom.

In some embodiments, when L is CH₂, S, C(O)NR⁶ or NR⁶C(O), R^(3a) is nota 5-membered heterocyclyl, 5-membered heteroaryl, or piperazine.

In some embodiments, when L is C(O)NH, R^(3a) and R^(3b) are not bothphenyl.

In some embodiments, m is at least 2 when L is connected to themethylene carbon via a heteroatom.

In some embodiments, R¹ is C₁-C₆ alkyl, for example, methyl.

In some embodiments, R¹ is further substituted by a dialkyl amine, forexample, a dimethyl amine.

In some embodiments, wherein R¹ is

In some embodiments, R¹ is C₁-C₆ alkyl substituted by heterocyclyl, forexample a nitrogen containing heterocyclyl such as morpholinyl.

In some embodiments, R² is C₁-C₆ alkyl, for example, methyl.

In some embodiments, R² is further substituted by a dialkyl amine, forexample, a dimethyl amine.

In some embodiments, wherein R² is

In some embodiments, R² is C₁-C₆ alkyl substituted by heterocyclyl, forexample, a nitrogen containing heterocyclyl such as morpholinyl.

In some embodiments, R^(3a) is monocyclic, for example a monocycliccyclyl, a monocyclic aryl, a monocyclic heterocyclyl, or a monocyclicheteroaryl.

In some embodiments, R^(3a) is aryl, for example, phenyl.

In some embodiments, R^(3a) is

In some embodiments, R^(3a) and/or R^(3b) is substituted by 1-4 R⁷.

In some embodiments, R^(3a) is heterocyclyl, for example, a nitrogencontaining heterocyclyl and/or a 5 membered heterocyclyl. In someembodiments, at least 1 R^(3b) is in the 3 position of the 5 memberedring.

In some embodiments, R^(3a) is substituted by 1 R⁷, for example, Me,OMe, or halo. In some embodiments, R⁷ is in a position other than the 3position of the 5 membered ring, for example, when R⁷ is Me, OMe, orhalo.

In some embodiments, R^(3a) is a 6 membered heterocyclyl, for example,R³ is

In some embodiments, R^(3a) and/or R^(3b) is substituted by 1-4 R⁷.

In some embodiments, R^(3a) is heteroaryl, for example, a 5 or 6membered heteroaryl, e.g., a 5 membered heteroaryl. In some embodiments,R^(3a) is substituted by 1-3 R⁷, for example, Me, OMe, or halo. In someembodiments, at least 1 R⁷ is in the 2 or 3 position of the 5 memberedring, for example when R⁷ is Me, OMe, or halo. In some embodiments, R³is substituted by 1 R⁷, for example, when R⁷ is Me, OMe, or halo. Insome embodiments, R⁷ is in the 2 or 3 position of the 5 membered ring,for example, when R⁷ is Me, OMe, or halo. In some embodiments, R⁷ is inthe 4 position of the 5 membered ring, for example, when R⁷ is Me, OMe,or halo.

In some embodiments, R^(3a) is a nitrogen containing heteroaryl, forexample,

In some embodiments, the 5 membered heteroaryl is substituted by atleast one R⁷ (e.g., one or two), for example, in the 3 or 4 position ofthe 5 membered ring. In some embodiments, R^(3a) and/or R^(3b) isfurther substituted by a cyclyl, heterocyclyl, aryl, heteroaryl (e.g.,phenyl, or thiophenyl, each of which is independently optionallysubstituted with 1-4 R⁷). In some embodiments, R^(3a) is

In some embodiments, R^(3b) is phenyl, optionally substituted by 1-4 R⁷.For example, 1 or 2 halo, Me, OMe, or amino.

In some embodiments, R^(3a) is a nitrogen containing heteroaryl, forexample,

In some embodiments, R^(3a) and/or R^(3b) is further substituted by 1-4R⁷.

In some embodiments, R^(3a) and R^(3b) together form

wherein R^(3a) and/or R^(3b) is optionally further substituted by 1-4R⁷. In some embodiments, the phenyl is further substituted by 1-3 R⁷. Insome embodiments, at least one R⁷ is amino, alkylamino, dialkylamino orheterocycyl (e.g., a nitrogen containing heterocyclyl such aspyrrolidine or piperidine). In some embodiments, at least 1 R⁷ ispositioned in the para position of the phenyl ring.

In some embodiments, R^(3a) is a 6 membered heteroaryl, for example,substituted by 1-3 R⁷ such as Me, OMe, or halo. In some embodiments, atleast 1 R⁷ is positioned in the para position, for example, when R⁷ isMe, OMe, or halo. In some embodiments, R^(3a) is substituted by 1 R⁷,for example, when R⁷ is Me, OMe, or halo. In some embodiments, R⁷ ispositioned in the para position, for example, when R⁷ is Me, OMe, orhalo.

In some embodiments, R^(3b) is a 6 membered, nitrogen containingheteroaryl.

In some embodiments, R^(3a) is

In some embodiments, R^(3a) is substituted by 1-4 R⁷.

In some embodiments, R^(3a) is a 6 membered heteroaryl containing 2nitrogens, e.g.,

In some embodiments, R^(3a) is substituted by 1-4 R⁷.

In some embodiments, R^(3a) is a heteroaryl or heterocycyl having twofused rings. In some embodiments, R^(3a) is substituted by 1-4 R⁷. Insome embodiments, R^(3a) is a heteroaryl or heterocycyl having threefused rings. In some embodiments, R^(3a) is substituted by 1-4 R⁷.

In some embodiments, R^(3b) is phenyl. In some embodiments, the phenylis further substituted with 1-3 R⁷.

In some embodiments, R^(3b) is a heteroaryl or heterocycyl having twofused rings. In some embodiments, R^(3b) is substituted by 1-4 R⁷.

In some embodiments, each R⁷ is independently C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, halo, hydroxyl, alkoxy, oxo, aryl, heteroaryl,cyclyl, heterocyclyl, aryloxy, arylalkoxy, amino, alkylamino,dialkylamino, thioyl, alkylthioyl, sulfonyl, sulfonamidyl, amido, urea,sulfonylurea, acyl, nitro, or cyano, each of which is optionallysubstituted with 1-3 R⁸.

In some embodiments, L is L is NR⁶SO₂, SO₂NR⁶, OC(O)NR⁶, NR⁶C(O)O,NR⁶C(O)NR⁶, S, S(O), S(O)₂, C(O)NS(O)₂, S(O)₂NC(O), heteroaryl, orcyclyl.

In some embodiments, L is NR⁶SO₂ or SO₂NR⁶, OC(O)NR⁶, NR⁶C(O)O,NR⁶C(O)NR⁶. In some embodiments, R⁶ is H.

In some embodiments, L is OC(O)NR⁶ or NR⁶C(O)O. In some embodiments, R⁶is H.

In some embodiments, L is NR⁶C(O)NR⁶. In some embodiments, R⁶ is H.

In some embodiments, L is cyclyl or heterocyclyl, for example,cyclopropyl.

In some embodiments, L is C(O)NR⁶ or NR⁶C(O). In some embodiments, R⁶ isH.

In some embodiments, L is C(O).

In some embodiments, R⁹ is H.

In some embodiments, R⁹ is halo, for example, chloro.

In some embodiments, m is 1.

In some embodiments, n is 2.

In some embodiments, m is 1 and n is 2, for example, where L is C(O)NR⁶.

In some embodiments, n is 0.

In some embodiments, m is 1 and n is 0, for example, where R³ is aryl orheteroaryl (e.g., further substituted by at least one R⁷).

In some embodiments, m+n≦6.

In some embodiments, the compound is of formula (VIII′)

Formula (VIII′). In some embodiments, L is C(O)NR⁶.

In one aspect, the compound is a compound of Formula (VIII″)

Formula (VIII″), wherein B is O, S, or NR⁶; D and E are independentlyCH, CR⁷ or N. In some embodiments, R^(3b) is phenyl, for example, aphenyl optionally substituted with 1-4 R⁷. In some embodiments, at leastone R⁷ is amino, alkylamino, dialkylamino or heterocycyl (e.g., anitrogen containing heterocyclyl such as pyrrolidine or piperidine). Insome embodiments, at least 1 R⁷ is positioned in the para position ofthe phenyl ring.

In some embodiments, D and E are independently O, S, or NR⁶.

In some embodiments, B is S, D is CH, and E is N.

In one aspect, the compound is a compound of Formula (VIII′″)

wherein B is O, S, or NR⁶; D and E are independently CH, CR⁷ or N. Insome embodiments, R^(3b) is phenyl, for example, a phenyl optionallysubstituted with 1-4 R⁷. In some embodiments, at least one R⁷ is amino,alkylamino, dialkylamino or heterocycyl (e.g., a nitrogen containingheterocyclyl such as pyrrolidine or piperidine). In some embodiments, atleast 1 R⁷ is positioned in the para position of the phenyl ring.

In some embodiments, D and E are independently O, S, or NR⁶.

In some embodiments, B is S, D is CH, and E is N.

In one aspect, the invention features a compound of formula (VIIIa)

wherein

R^(3a) cyclyl, heterocyclyl, aryl, heteroaryl,

R^(3b) is cyclyl, heterocyclyl, aryl, heteroaryl; optionally substitutedwith 1-3 R⁷;

each R⁷ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, oxo, hydroxyl, alkoxy, aryl, heteroaryl, cyclyl, heterocyclyl,aryloxy, arylalkoxy, amino, alkylamino, dialkylamino, thioyl,alkylthioyl, sulfonyl, sulfonamidyl (e.g., where the nitrogen of thesulfonamide is substituted by an alkyl, or where the nitrogen of thesulfonamide together with two carbons to which it is attached, forms aring), amido(e.g., where the nitrogen of the amide is substituted by analkyl, or where the nitrogen of the amide together with two carbons towhich it is attached, forms a ring), hydroxyl alkoxyl, alkoxy alkoxyl,urea, sulfonylurea, acyl, nitro, cyano, each of which is optionallysubstituted with 1-3 R⁸;

each R⁸ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,aryl, heteroaryl, cyclyl, halo, hydroxyl, alkoxy, aryloxy, amino,alkylamino, dialkylamino, thioyl, sulfonyl, sulfonamidyl, amido, urea,sulfonylurea acyl, nitro, cyano, cyclyl, heterocyclyl, aryl, orheteroaryl; and

R⁹ is H or halo.

In some embodiments, R^(3a) is aryl or heteroaryl.

In some embodiments, R^(3a) is heteroaryl.

In some embodiments, R^(3a) is thiazoyl.

In some embodiments, R⁷ is C₁-C₆ alkyl, halo, aryl, or heteroaryl, forexample, optionally substituted with 1-3 R⁸

In some embodiments, R^(3b) is aryl or heteroaryl.

In one aspect, the invention features a compound of formula (VIIb)

wherein R^(3b) is aryl or heteroaryl; optionally substituted with 1-3R⁷;

each R⁷ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,halo, hydroxyl, alkoxy, aryl, heteroaryl, cyclyl, heterocyclyl, aryloxy,arylalkoxy, amino, alkylamino, dialkylamino, thioyl, alkylthioyl,sulfonyl, sulfonamidyl, amido, hydroxyl alkoxyl, alkoxy alkoxyl, urea,sulfonylurea, acyl, nitro, cyano, each of which is optionallysubstituted with 1-3 R⁸;

R^(7a) is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, halo,hydroxyl, alkoxy, aryl, heteroaryl, cyclyl, heterocyclyl, aryloxy,arylalkoxy, amino, alkylamino, dialkylamino, thioyl, alkylthioyl,sulfonyl, sulfonamidyl, amido, hydroxyl alkoxyl, alkoxy alkoxyl, urea,sulfonylurea, acyl, nitro, cyano, each of which is optionallysubstituted with 1-3 R⁸;

each R⁸ is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl,aryl, heteroaryl, cyclyl, halo, hydroxyl, alkoxy, aryloxy, amino,alkylamino, dialkylamino, thioyl, sulfonyl, sulfonamidyl, amido, urea,sulfonylurea acyl, nitro, cyano, cyclyl, heterocyclyl, aryl, orheteroaryl; and

R⁹ is H or halo.

In some embodiments, R^(3b) is phenyl.

In some embodiments, R^(3b) is phenyl substituted with at least 1 R⁷ andwherein at least 1 R⁷ is amino, alkylamino, dialkylamino or heterocycyl(e.g., a nitrogen containing heterocyclyl such as pyrrolidine orpiperidine).

In some embodiments, R^(3b) is

In some embodiments, R^(3b) is further substituted by at least oneadditional R⁷ (e.g., a halo).

In some embodiments, R⁷ is amino, alkylamino, dialkylamino orheterocycyl (e.g., a nitrogen containing heterocyclyl such aspyrrolidine or piperidine).

In some embodiments, R⁷ is diethylamino.

In some embodiments, R⁷ is pyrrolidinyl.

In some embodiments, R^(3b) is further substituted by at least oneadditional R⁷ (e.g., a halo).

In some embodiments, at least 1 R⁷ is positioned in the para position ofthe phenyl ring.

In some embodiments, R^(3b) is a bicyclic fused aryl or heteroaryl, forexample, optionally substituted with 1-3 R⁷.

In some embodiments, R⁷ is H.

In some embodiments, R⁷ is C₁-C₆ alkyl.

In some embodiments, R⁹ is H.

In some embodiments, a compound is a substantially pure stereoisomer.For example, in some embodiments, a compound described herein has beenpurified to provide a substantially chiraly enriched compound (e.g.,wherein the compound is substantially free of other stereoisomers). Insome embodiments, the compound is at least about 60% pure, e.g., atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 97%, at least about 98%, or at least about 99%.

In some embodiments, the compound is not a compound depicted in Table 2(e.g., is not any one of the individual compounds) having a formuladescribed herein. For example, the compounds of the invention are not acompound described in Table 2 of U.S. Ser. No. 11/645,307 filed Dec. 12,2006, which is incorporated herein by reference in its entirety.

One aspect of the present invention provides a pharmaceuticalpreparation suitable for use in a human patient, or for veterinary use,comprising an effective amount of any of the compounds shown above(e.g., a compound of described herein or a salt thereof, or a solvate,hydrate, oxidative metabolite or prodrug of the compound or its salt),and one or more pharmaceutically acceptable excipients. In certainembodiments, the pharmaceutical preparations may be for use in treatingor preventing a condition involving activation of TRPA1 or for whichreduced TRPA1 activity can reduce the severity. In certain embodiments,the pharmaceutical preparations have a low enough pyrogen activity to besuitable for use in a human patient, or for veterinary use. In certainembodiments, the pharmaceutical preparation comprises an effectiveamount of any of the compounds shown above, wherein the compoundinhibits TRPA1 (e.g., a TRPA1-mediated current and/or TRPA1-mediated ionflux) with an IC₅₀ of 10 micromolar or less. In certain embodiments, thepharmaceutical preparation comprises a compound which inhibits TRPA1with an IC₅₀ of 5 micromolar or less, 2 micromolar or less, 1 micromolaror less, or even with an IC₅₀ of 500 nM or less, 250 nM or less, 200 nMor less, or even 100 nM or less.

TRPA1 antagonists of the subject invention can be used as part of aprophylaxis or treatment for a variety of disorders and conditions,including, but not limited to, acute and/or chronic pain, touchsensitivity, burns, inflammation, diabetic neuropathy, psoriasis,eczema, dermatitis, post-herpetic neuralgia (shingles), migraine,incontinence, fever, hot flashes, osteoarthritis, oral mucositis, cancerpain, bladder cystits, pain associated with Crohn's disease andIrritable Bowel Syndrome (IBS), rheumatoid arthritis, Grierson-Gopalansyndrome (better known as burning feet syndrome), burning mouth syndrome(BMS) and cough, or is used as a depilatory to promote loss of orinhibit the growth of hair on a patient. Other exemplary diseases orconditions that can be treated using a TRPA1 antagonist of the presentinvention are detailed throughout the specification. The inventioncontemplates the use of compounds having any of the structures providedin the specification in the treatment of or to reduce the symptoms ofany of the diseases or conditions disclosed in the application. Theinvention further contemplates the use of compounds having any of thestructures provided in the specification in the manufacture of amedicament or pharmaceutical preparation to treat or reduce the symptomsof any of the diseases or conditions provided in the specification.Compounds for use in treating a particular disease or condition can beformulated for administration via a route appropriate for the particulardisease or condition.

TRPA1 antagonists can be administered alone or in combination with othertherapeutic agents. For instance, the TRPA1 antagonists is administeredconjointly with one or more of an anti-inflammatory agent, anti-acneagent, anti-wrinkle agent, anti-scarring agent, anti-psoriatic agent,anti-proliferative agent, anti-fungal agent, anti-viral agent,anti-septic agent, anti-migraine agent, keratolytic agent, or a hairgrowth inhibitor.

TRPA1 antagonists can be administered topically, orally, transdermally,rectally, vaginally, parentally, intranasally, intrapulmonary,intraocularly, intravenously, intramuscularly, intraarterially,intrathecally, intracapsularly, intraorbitally, intracardiacly,intradermally, intraperitoneally, transtracheally, subcutaneously,subcuticularly, intraarticularly, subcapsularly, subarachnoidly,intraspinally, intrasternally or by inhalation.

In certain preferred embodiments, a TRPA1 antagonist is administeredtopically.

In certain preferred embodiments, a TRPA1 antagonist is administeredorally.

In certain preferred embodiments, a TRPA1 antagonist is administeredparentally.

In certain preferred embodiments, a TRPA1 antagonist is administered toprevent, treat or alleviate signs and symptoms of acute pain, chronicpain, touch sensitivity, itching sensitivity, or as part of treating aburn, such as, for example, post-surgical pain, cancer pain, orneuropathic pain.

In certain preferred embodiments, a TRPA1 antagonist is administered toprevent, treat or alleviate signs and symptoms of migraine.

In certain preferred embodiments, a TRPA1 antagonist is administered toprevent, treat or alleviate signs and symptoms of a disorder orcondition selected from the group consisting of diabetic neuropathy,inflammation, psoriasis, eczema, dermatitis, post-herpetic neuralgia(shingles), incontinence, bladder incontinence, fever, hot flashes,pancreatitis, chronic regional pain syndrome, Fabray's disease, andcough.

In certain preferred embodiments, a TRPA1 antagonist is administered toprevent, treat or alleviate signs and symptoms of osteoarthritis.

In certain preferred embodiments, a TRPA1 antagonist is administered toprevent, treat or alleviate signs and symptoms of rheumatoid arthritis.

In certain preferred embodiments, a TRPA1 antagonist is administered toprevent, treat or alleviate signs and symptoms of oral mucositis.

In certain preferred embodiments, a TRPA1 antagonist is administered topromote loss of or inhibit the growth of hair on a patient.

Still another aspect of the present invention relates to the use of aTRPA1 antagonist, e.g., a small molecule agent that inhibits inwardTRPA1-mediated current with an IC₅₀ of 1 micromolar or less, in themanufacture of a medicament to prevent, treat or alleviate symptoms of adisease, disorder or condition involving activation of TRPA1, or forwhich reduced TRPA1 activity can reduce the severity, in a patient.

Yet another aspect of the present invention relates to a pharmaceuticalpreparation comprising an agent that inhibits inward TRPA1-mediatedcurrent with an IC₅₀ of 1 micromolar or less; and a pharmaceuticallyacceptable excipient or solvent wherein the agent is provided in adosage form providing an amount effective to prevent, treat or alleviatesymptoms of a disease, disorder or condition involving activation ofTRPA1, or for which reduced TRPA1 activity can reduce the severity, in apatient. In certain preferred embodiments, the pharmaceuticalpreparation does not cause QT interval elongation in the patient.

In certain illustrative embodiments, the pharmaceutical preparationcomprises an agent that inhibits TRPA1-mediated current with an IC₅₀ ofat least one order of magnitude lower than its IC₅₀ for inhibition ofNaV 1.2 function, TRPV1 function, TRPV5 function, TRPV6 function,mitochondrial uniporter function and HERG function; and apharmaceutically acceptable excipient or solvent, wherein the agent isprovided in a dosage form providing an amount effective to prevent,treat or alleviate symptoms of a disease, disorder or conditioninvolving activation of TRPA1, or for which reduced TRPA1 activity canreduce the severity, in a patient, but which does not cause QT intervalelongation.

In another illustrative embodiment, the pharmaceutical preparationcomprises an agent that inhibits a TRPA1-mediated current with an IC₅₀of 1 micromolar or less; and a pharmaceutically acceptable excipient orsolvent, wherein the agent is provided in a dosage form providing anamount effective to prevent, treat or alleviate symptoms of a disease,disorder or condition involving activation of TRPA1, or for whichreduced TRPA1 activity can reduce the severity, in a patient, but whichdoes not cause QT interval elongation.

One preferred preparation is a topical formulation for reducing TRPA1activity in skin or mucosa, comprising an agent that inhibits aTRPA1-mediated current with an IC₅₀ of 1 micromolar or less.

Another preferred preparation is a removable patch or bandage,comprising: (i) a polymeric base; and (ii) an agent that inhibits aTRPA1-mediated current with an IC₅₀ of 1 micromolar or less.

Still another illustrative formulation is a skin exfoliant compositionfor topical application to an animal subject comprising a topicalvehicle; one or more skin exfoliant ingredients selected from the groupconsisting of carboxylic acids, keto acids, α-hydroxy acids, β-hydroxyacids, retinoids, peroxides, and organic alcohols, said one or more skinexfoliant ingredients contained in a total amount of at least about 12%by weight and capable of inducing skin irritation and effectingexfoliation of the skin of said subject; and an agent that inhibits aTRPA1-mediated current with an IC₅₀ of 1 micromolar or less, which agentis provided in an amount effective for analgesic, anti-irritant and/oranti-inflammatory effects when applied to skin.

Yet another embodiment is an antitussive composition for peroraladministration comprising an agent that inhibits both a TRPA1-mediatedcurrent with an IC₅₀ of 1 micromolar or less, and an orally-acceptablepharmaceutical carrier in the form of an aqueous-based liquid, or soliddissolvable in the mouth, selected from the group consisting of syrup,elixer, suspension, spray, lozenge, chewable lozenge, powder, andchewable tablet. Such antitussive compositions can include one or moreadditional agents for treating cough, allergy or asthma symptom selectedfrom the group consisting of: antihistamines, 5-lipoxygenase inhibitors,leukotriene inhibitors, H3 inhibitors, β-adrenergic receptor agonists,xanthine derivatives, α-adrenergic receptor agonists, mast cellstabilizers, expectorants, NK1, NK2 and NK3 tachykinin receptorantagonists, and GABA_(B) agonists.

Still another embodiment is a metered dose aerosol dispenser containingan aerosol pharmaceutical composition for pulmonary or nasal deliverycomprising an agent that inhibits a TRPA1-mediated current with an IC₅₀of 1 micromolar or less. For instance, it can be a metered dose inhaler,a dry powder inhaler or an air-jet nebulizer.

Still another embodiment is an eye ointment or eyedrops for ocularadministration. Such ocular compositions may be useful for the treatmentor alleviation of ocular pain including pain resulting from eye abrasionor post-surgical pain.

In another aspect, the invention contemplates that any of the TRPA1inhibitors of the present invention, including inhibitors having one ormore of the characteristics disclosed herein, can be used to inhibit afunction of TRPA1, for example a TRPA1-mediated current and/or aTRPA1-mediated ion flux. In some embodiments, the compounds can be usedto inhibit a TRPA1 mediated current in vitro, for example in cells inculture. In some embodiments, the compounds can be used to inhibit aTRPA1 mediated current in vivo. In certain embodiments, the compoundsinhibit both an inward and an outward TRPA1-mediated current. In certainembodiments, the compounds inhibit a TRPA1 mediated ion flux in vitro,for example in cells in culture. In certain other embodiments, thecompounds inhibit a TRPA1 mediated in flux in vivo.

The invention contemplates pharmaceutical preparations and uses of TRPA1antagonists having any combination of the foregoing or followingcharacteristics, as well as any combination of the structural orfunctional characteristics of the TRPA1 antagonists described herein.Any such antagonists or preparations can be used in the treatment of anyof the diseases or conditions described herein. Any such antagonists orpreparations can be used to inhibit a function of TRPA1, for example aTRPA1-mediated current and/or a TRPA1-mediated ion flux.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are graphs showing the plasma levels of compounds 7(intravenous or oral administration) and 8 (oral administration) ofTable 3 over time.

FIG. 1C is a table summarizing the pharmacokinetics of compound 7 ofTable 3 following oral and intravenous administration.

FIGS. 2A and 2B are graphs showing the plasma levels of compound 7 andcompound 8 of Table 3 over time following oral administration.

FIG. 3 is a graph showing the total number of flinches in the variousphases of the pain response in rats subjected to the formalin test.

FIG. 4 is a graph showing the latency in rats treated with gapapentin orcompound 7 of Table 3.

FIG. 5 is Table 2, showing compounds that are not part of the invention.

FIG. 6 is Table 4 a table showing exemplary compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Cellular homeostasis is a result of the summation of regulatory systemsinvolved in, amongst other things, the regulation of ion flux andmembrane potential. Cellular homeostasis is achieved, at least in part,by movement of ions into and out of cells across the plasma membrane andwithin cells by movement of ions across membranes of intracellularorganelles including, for example, the endoplasmic reticulum,sarcoplasmic reticulum, mitochondria and endocytic organelles includingendosomes and lysosomes.

Movement of ions across cellular membranes is carried out by specializedproteins. TRP channels are one large family of non-selective cationchannels that function to help regulate ion flux and membrane potential.TRP channels are subdivided into 6 sub-families including the TRPA(ANKTM1) family. TRPA1 is a member of the TRPA class of TRP channels.

Non-selective cation channels such as TRPA1 modulate the flux of calciumand sodium ions across cellular membranes. Sodium and calcium influxleads to a depolarization of the cell. This increases the probabilitythat voltage-gated ion channels will reach the threshold required foractivation. As a result, activation of non-selective cation channels canincrease electrical excitability and increase the frequency ofvoltage-dependent events. Voltage-dependent events include, but are notlimited to, neuronal action potentials, cardiac action potentials,smooth muscle contraction, cardiac muscle contraction, and skeletalmuscle contraction.

Calcium influx caused by the activation of non-selective cation channelssuch as TRPA1 also alters the intracellular free calcium concentration.Calcium is a ubiquitous second messenger molecule within the cell. Thusalterations in intracellular calcium levels have profound effects onsignal transduction and gene expression. Thus, activation ofnon-selective cation channels such as TRPA1 can lead to changes in geneexpression and cellular phenotype. Gene expression events include, butare not limited to, production of mRNAs encoding cell surface receptors,ion channels, and kinases. These changes in gene expression can lead tohyperexcitability in that cell. Blockers of TRPA1 therefore also havethe potential to decrease or prevent pain and/or to decrease overactivebladder.

TRPA1 proteins are receptor operated channels expressed in sensoryneurons (see, e.g., Jordt et al. (2004) Nature 427:260-265) includingthose with cell bodies residing in the dorsal root ganglion, trigeminalganglion, and nodose ganglia (see Jordt et al. (2004) Nature427:260-265, Nagata et al. (2005) J. Neurosci 25(16) 4052-61). Inaddition, low levels of TRPA1 message can be found in some types offibroblasts (see Jaquemar et al. (1999) JBC 274(11): 7325-33). TRPA1 hasalso been reported to be expressed in the bladder. Stimulation of anumber of extracellular receptors, including, but not limited to,G-protein coupled receptors or receptor tyrosine kinases are sufficientto activate TRPA1.

TRPA1 proteins suitable for use in accordance with the methods providedherein include, for example: human (SEQ ID NO: 1 and SEQ ID NO: 3 aminoacid sequences, encoded by SEQ ID NO: 2 and SEQ ID NO: 4 nucleotidesequences respectively) and murine (SEQ ID NO: 5 amino acid sequence,encoded by SEQ ID NO: 6 nucleotide sequence). Particular TRPA1 proteinsalso include proteins encoded by cDNAs that would hybridize to the TRPA1sequence (see SEQ ID NO: 2) under stringent conditions.

TRPA1 is the ion channel that responds to mustard oil. The activeingredients in mustard oil (allyl isothiocyanate) and the activeingredient in garlic (allicin) are both capable of activating TRPA1.Other stimuli may also be able to activate TRPA1. It has been reportedthat severe cold temperatures between 4 and 15° C. activate TRPA1 (seeStory et al., (2003) Cell 112(6): 819-829). However, this finding hasbeen controversial (see Jordt et al. (2004) Nature 427:260-265; Nagataet al. (2005) J. Neurosci 25(16): 4052-61). In addition, TRPA1 sharesmany structural similarities with TRP channels (i.e., TRPN1, DrosophilaTRPA1) in lower animals that respond to mechanical stimulation.

TRPA1 is expressed in, among other tissues, the hair cell epithelia ofthe inner ear, and disruption of this channel in zebrafish and mouseinhibits hair cell transduction. Therefore, TRPA1 has been proposed inthe art as a candidate for the mechanosensitive vertebrate hearingtransduction channel (see Corey et al., (2004) Nature 432(7018):723-730). If this were the case, it would suggest that blockers of TRPA1might lead to hearing loss, and thus would not have any practical use asa therapeutic agent. However, the observation that the startle responseis not substantially impaired in the TRPA1 knockout mouse has led us toconclude that TRPA1 antagonists may not impair hearing, and would thusbe suitable drug candidates.

Modulating the function of TRPA1 proteins provides a means of modulatingcalcium homeostasis, sodium homeostasis, membrane polarization, and/orintracellular calcium levels, and compounds that can modulate TRPA1function are useful in many aspects, including, but not limited to,maintaining calcium homeostasis, modulating intracellular calciumlevels, modulating membrane polarization, and treating or preventingdiseases, disorders, or conditions associated with calcium and/or sodiumhomeostasis or dyshomeostasis.

In certain aspects, the present invention provides methods for treatingor ameliorating the effects of diseases and conditions using smallmolecules that inhibit a TRPA1-mediated current and/or a TRPA1-mediatedion flux with an IC₅₀ of less than 10 micromolar. Exemplary suitablecompounds for use in any of the methods of the invention (e.g., to treatany of the diseases or conditions disclosed herein) include compoundshaving one or more of the structural or functional characteristicsdisclosed herein (e.g., structure, specificity, potency, solubility,etc.).

The present invention contemplates the use of any TRPA1 antagonistpossessing one or more of the functional or structural attributesdescribed herein. Additionally, the present invention contemplates theuse of TRPA1 antagonists of a compound described herein, as well as theuse of any of the particular antagonists provided in Tables 1, 3 or 4.Throughout the application, when particular functional attributes areattributed to TRPA1 antagonists, it is understood that such attributesmay characterize TRPA1 inhibitors structurally related to or differingfrom a compound described herein.

In certain embodiments, a suitable compound inhibits an inward and/oroutward TRPA1 mediated current with an IC₅₀ of less than 10 micromolar.In certain embodiments, a suitable compound additionally oralternatively inhibits TRPA1 mediated ion flux with an IC₅₀ of less than10 micromolar. IC₅₀ can be calculated, for example, in an in vitroassay. For example, IC50 can be calculated using electrophysiologicaldeterminations of current, such as standard patch clamp analysis. IC₅₀can also be evaluated using changes in concentration or flux of ionindicators, such as the calcium flux methods described herein.

In certain embodiments, the invention provides a method for treating orpreventing a condition involving activation of TRPA1 or for whichreduced TRPA1 activity can reduce the severity, comprising administeringan effective amount of a compound described herein or a salt thereof, ora solvate, hydrate, oxidative metabolite or prodrug of the compound orits salt:

Exemplary compounds are provided in Tables 1, 3, and 4:

A represents a compound demonstrating activity of <1 μM as measured inthe patch clamp assay. B represents a compound demonstrating activityof >1 μM-<10 μM as measured in the patch clamp assay. C represents acompound demonstrating activity of >10 μM as measured in the patch clampassay. D represents other exemplary compounds.

TABLE 1 1 2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(3-methoxyphenyl)acetamide 22-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- C(4-fluorophenyl)acetamide 32-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- C7(6H)-yl)-N-p-tolylacetamide 4(E)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)- BN′-(4-(trifluoromethyl)benzylidene)acetohydrazide 52-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- Dmethyl-N-(2-(pyridin-2-yl)ethyl)acetamide 62-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(2-(pyridin-2-yl)ethyl)acetamide 72-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(3-methoxyphenyl)acetamide 82-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- A(2-adamantylethyl)acetamide 92-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- B(5-fluoro-2-methylphenyl)acetamide 10N-cyclooctyl-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)acetamide 112-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-(adamantylmethyl)acetamide 122-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(thiochroman-4-yl)acetamide 132-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(2-fluorophenyl)acetamide 142-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(2-(furan-2-yl)-2-(pyrrolidin-1-yl)ethyl)acetamide 152-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-(3-fluoro-4-methoxybenzyl)-N-methylacetamide 162-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-(3-methoxybenzyl)acetamide 172-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-(4-fluorophenethyl)acetamide 182-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-p- Atolylpropanamide 19N-((2,3-dihydrobenzo[b][1,4]dioxin-2-yl)methyl)-2-(1,3- Ddimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 202-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- A7(6H)-yl)-N-(2,3-dihydro-1H-inden-5-yl)acetamide 212-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-(2,3-dimethylcyclohexyl)acetamide 22 methyl4-((2-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H- Dpurin-7(6H)-yl)acetamido)methyl)benzoate 23N-(3,4-dimethoxybenzyl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Ddihydro-1H-purin-7(6H)-yl)acetamide 242-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-isobutyl-N-(dioxytetrahydrothiophen-3- yl)acetamide 25N-((3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methyl)-2-(1,3- Ddimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- methylacetamide 262-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-methyl-N-(4-methylbenzyl)acetamide 272-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-cyclohexyl-N-ethylacetamide 282-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-(3,4-dihydro-2H-benzo[b][1,4]dioxepin-7- yl)acetamide 292-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-(1-cyclopentyl-1H-pyrazol-5-yl)acetamide 302-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-methyl-N-(1,2,3,4-tetrahydronaphthalen-1- yl)acetamide 312-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-(4-phenylbutan-2-yl)acetamide 32N-cyclohexyl-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-ethylacetamide 332-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- A7(6H)-yl)-N-(2-(4-chlorophenylthio)ethyl)acetamide 34N-((2,3-dihydrobenzo[b][1,4]dioxin-2-yl)methyl)-2-(1,3- Ddimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- methylacetamide 35N-(2-cyclohexenylethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro- D1H-purin-7(6H)-yl)acetamide 362-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-(1-(5,6,7,8-tetrahydronaphthalen-2- yl)ethyl)acetamide 37N-(3,5-dichloropyridin-2-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Ddihydro-1H-purin-7(6H)-yl)propanamide 38N-(5-chloropyridin-2-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro- D1H-purin-7(6H)-yl)propanamide 39N-((4-chlorophenyl)(cyclopropyl)methyl)-2-(1,3-dimethyl-2,6- Ddioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 40N-(1-(3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl)-2- Dmethylpropyl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 41N-((1-benzyl-1H-pyrazol-4-yl)methyl)-2-(1,3-dimethyl-2,6- Ddioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-methylacetamide 422-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- C(2,2-diphenylpropyl)acetamide 432-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D((2-ethylbenzofuran-3-yl)methyl)-N-methylacetamide 44N-(cyclohexylmethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro- B1H-purin-7(6H)-yl)acetamide 45N-cyclohexyl-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-methylpropanamide 463-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- C(4-methoxybenzyl)propanamide 472-(1,3-diethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(4- Cmethoxyphenethyl)acetamide 481-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- C(4-methoxyphenethyl)methanesulfonamide 49N-(2-cyclohexylethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro- C1H-purin-7(6H)-yl)acetamide 502-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- C(2-(4-hydroxycyclohexyl)ethyl)acetamide 512-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- B(3,4-dimethylphenethyl)acetamide 52N-(2-(biphenyl-4-yl)ethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Adihydro-1H-purin-7(6H)-yl)acetamide 53N-(4-(benzyloxy)phenethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Bdihydro-1H-purin-7(6H)-yl)acetamide 542-(1,3-dimethyl-2,6,8-trioxo-2,3,7,8-tetrahydro-1H-purin-9(6H)- Cyl)-N-(4-methoxyphenethyl)acetamide 552-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- C((3R,4R)-quinuclidin-3-yl)acetamide 56N-(4-butoxyphenethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro- B1H-purin-7(6H)-yl)acetamide 57N-(4-cyclohexylphenethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Adihydro-1H-purin-7(6H)-yl)acetamide 582-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- B((1S,2R)-2-(4-methoxyphenyl)cyclopropyl)acetamide 59N-(4-tert-butylphenethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Bdihydro-1H-purin-7(6H)-yl)acetamide 602-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- B(4-methoxybenzylsulfonyl)acetamide 61N-(4-methoxyphenethyl)-2-(1,3,9-trimethyl-2,6,8-trioxo-2,3- Bdihydro-1H-purin-7(6H,8H,9H)-yl)acetamide 62 4-methylphenethyl2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H- B purin-7(6H)-yl)acetate 632-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- C(2-(piperidin-1-yl)ethyl)acetamide 642-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- C(2-morpholinoethyl)acetamide 652-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- B((1S,2S)-2-(4-methoxyphenyl)cyclopropyl)acetamide 662-(1,3-dicyclopropyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)- DN-(4-methoxyphenethyl)acetamide 672-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(2-(4-methoxycyclohexyl)ethyl)acetamide 682-(1,3-dimethyl-2,6,8-trioxo-2,3-dihydro-1H-purin- C7(6H,8H,9H)-yl)-N-(4-methoxyphenethyl)acetamide 692-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- C(2-(pyridin-4-yl)ethyl)acetamide 702-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- B(2-(4-methylpiperazin-1-yl)ethyl)acetamide 711,3-dimethyl-7-(2-(4-methylphenethylamino)ethyl)-1H-purine- B2,6(3H,7H)-dione 72N-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)- Byl)ethyl)-N-(4-methylphenethyl)methanesulfonamide 732-(3-methyl-2,6-dioxo-1-propyl-2,3-dihydro-1H-purin-7(6H)- Cyl)-N-(4-methylphenethyl)acetamide 741,3-dimethyl-7-(2-(methyl(4-methylphenethyl)amino)ethyl)-1H- Bpurine-2,6(3H,7H)-dione 752-(1-methyl-2,6-dioxo-3-propyl-2,3-dihydro-1H-purin-7(6H)- Byl)-N-(4-methylphenethyl)acetamide 76N-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)- Ayl)ethyl)-N-(4-methylphenethyl)acetamide 772-(1-methyl-2,6-dioxo-3-propyl-2,3-dihydro-1H-purin-7(6H)- Ayl)-N-phenethylacetamide 782-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- B(2-phenylpropyl)acetamide 792-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- B(3-fluorophenethyl)acetamide 802-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- B(4-ethylphenethyl)acetamide 81(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)- BN-(1-hydroxy-3-phenylpropan-2-yl)acetamide 82N-(2,3-dimethoxyphenethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Bdihydro-1H-purin-7(6H)-yl)acetamide 83N-(2,3-dichlorophenethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Bdihydro-1H-purin-7(6H)-yl)acetamide 84N-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-2-(1,3-dimethyl-2,6- Bdioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 85(R)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)- BN-(1-hydroxy-3-phenylpropan-2-yl)acetamide 86N-(2,5-dimethoxyphenethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Bdihydro-1H-purin-7(6H)-yl)acetamide 87N-(2,4-dichlorophenethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Bdihydro-1H-purin-7(6H)-yl)acetamide 88N-(2,6-dichlorophenethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Bdihydro-1H-purin-7(6H)-yl)acetamide 89N-(2-bromophenethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro- B1H-purin-7(6H)-yl)acetamide 902-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(2-(quinuclidin-3-yl)ethyl)acetamide 912-(3-(2-(dimethylamino)ethyl)-1-methyl-2,6-dioxo-2,3-dihydro- D1H-purin-7(6H)-yl)-N-(4-methylphenethyl)acetamide 922-(1-(2-(dimethylamino)ethyl)-3-methyl-2,6-dioxo-2,3-dihydro- D1H-purin-7(6H)-yl)-N-(4-methylphenethyl)acetamide 93N-(6-chlorobenzo[d]thiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Ddihydro-1H-purin-7(6H)-yl)acetamide 94N-(6-bromobenzo[d]thiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Adihydro-1H-purin-7(6H)-yl)acetamide 95N-(4-chlorobenzo[d]thiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Ddihydro-1H-purin-7(6H)-yl)acetamide 962-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(5,6-dimethylbenzo[d]thiazol-2-yl)acetamide 972-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- A(6-ethoxybenzo[d]thiazol-2-yl)acetamide 982-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(4-methoxybenzo[d]thiazol-2-yl)acetamide 992-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(4-methylbenzo[d]thiazol-2-yl)acetamide 1002-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(6-(methylsulfonyl)benzo[d]thiazol-2-yl)acetamide 1012-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(2-hydroxy-2-phenylethyl)acetamide 102N-(3-chlorophenethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro- D1H-purin-7(6H)-yl)acetamide 103(S)-2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)- Dyl)acetamido)-3-phenylpropanamide 104N-(2-chloro-6,7-dimethoxyquinazolin-4-yl)-2-(1,3-dimethyl-2,6- Ddioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 105N-(4-bromophenethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro- D1H-purin-7(6H)-yl)acetamide 1062-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(2,2-diphenylethyl)acetamide 107 (S)-methyl2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)acetamido)-3-phenylpropanoate 108N-(3,5-dimethoxyphenethyl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Ddihydro-1H-purin-7(6H)-yl)acetamide 109(R)-2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)- Dyl)acetamido)-3-phenylpropanamide 1102-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)acetamide 111 ethyl2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)- Dyl)acetamido)-5-methylthiazole-4-carboxylate 112N-(2-(1,4-diazabicyclo[2.2.2]octan-2-yl)ethyl)-2-(1,3-dimethyl- D2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 113 ethyl2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)- Dyl)acetamido)benzo[d]thiazole-6-carboxylate 1142-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- Dmethyl-N-phenethylacetamide 1151,3-dimethyl-7-((5-(4-methylbenzyl)-1,3,4-oxadiazol-2- Dyl)methyl)-1H-purine-2,6(3H,7H)-dione 1162-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(2-(6-methylpyridin-3-yl)ethyl)acetamide 1172-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(2-(1-methyl-1H-imidazol-2-yl)ethyl)acetamide 1181,3-dimethyl-7-(3-(6-methyl-1H-indol-2-yl)propyl)-1H-purine- D2,6(3H,7H)-dione 1192-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- Dethyl-N-(pyridin-4-ylmethyl)acetamide 120N-(4,5-dihydrothiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Ddihydro-1H-purin-7(6H)-yl)acetamide 121 ethyl2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)- Dyl)acetamido)-4-methylthiazole-5-carboxylate 122N-(4H-chromeno[4,3-d]thiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo- D2,3-dihydro-1H-purin-7(6H)-yl)acetamide 123 ethyl2-(2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)acetamido)thiazol-4-yl)acetate 1242-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(4-methylthiazol-2-yl)acetamide 1252-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(1,2,3,4-tetrahydronaphthalen-1-yl)acetamide 126N-(2,3-dihydro-1H-inden-1-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Ddihydro-1H-purin-7(6H)-yl)acetamide 127N′-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)- Dyl)acetyl)-4-methylbenzohydrazide 1282-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(2-(5-methylpyridin-2-yl)ethyl)acetamide 1291-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)- Dyl)ethyl)-1-(4-methylphenethyl)urea 1301,3-dimethyl-7-((5-p-tolyl-1,3,4-oxadiazol-2-yl)methyl)-1H- Dpurine-2,6(3H,7H)-dione 131N-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)- Dyl)ethyl)-N-(4-methylphenethyl)propionamide 1321,3-dimethyl-7-((2-(4-methylphenethyl)cyclopropyl)methyl)-1H- Dpurine-2,6(3H,7H)-dione 133N-(3-bromo-4-methoxyphenethyl)-2-(1,3-dimethyl-2,6-dioxo- D2,3-dihydro-1H-purin-7(6H)-yl)acetamide 134N-((1R,2R)-1,3-dihydroxy-1-phenylpropan-2-yl)-2-(1,3- Ddimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 1352-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(piperidin-4-yl)acetamide 1362-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(2,2,6,6-tetramethylpiperidin-4-yl)acetamide 1372-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(2-hydroxy-2-phenylethyl)-N-methylacetamide 1382-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(4,6-dimethylpyrimidin-2-yl)acetamide 139N-benzyl-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin- D7(6H)-yl)-N-(4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)acetamide 140N-benzyl-2-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H- Dpurin-7(6H)-yl)-N-(4,5,6,7-tetrahydrobenzo[d]thiazol-2- yl)acetamide 141N-(4-acetylthiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro- D1H-purin-7(6H)-yl)acetamide 1422-(8-chloro-1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)-N-(4-methylphenethyl)acetamide; 143N-(6-butylbenzo[d]thiazol-2-yl)-2-(8-chloro-1,3-dimethyl-2,6- Ddioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide; 1442-(8-chloro-1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)-N-(2-(adamant-1-yl)ethyl)acetamide; 1451-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethyl)-1-(4-methylphenethyl)urea; 146N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethyl)-N-(4-methylphenethyl)propionamide; 147N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethyl)-N-(4-methylphenethyl)pentanamide; 1482-(3-(2-hydroxyethyl)-1-methyl-2,6-dioxo-1,2,3,6- Dtetrahydropurin-7-yl)-N-(4-methylphenethyl)acetamide; 1492-(3-(2-amino-2-oxoethyl)-1-methyl-2,6-dioxo-1,2,3,6- Dtetrahydropurin-7-yl)-N-(4-methylphenethyl)acetamide; 150N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethyl)-4-oxo-4-phenylbutanamide; 151N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethyl)-5-(4-fluorophenyl)-5-oxopentanamide; 1522-(3-chloro-4-(trifluoromethyl)phenyl)-N-(2-(1,3-dimethyl-2,6- Ddioxo-1,2,3,6-tetrahydropurin-7-yl)ethyl)acetamide; 1532-(3-chloro-4-fluorophenyl)-N-(2-(1,3-dimethyl-2,6-dioxo- D1,2,3,6-tetrahydropurin-7-yl)ethyl)acetamide; 1541-benzyl-N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin- D7-yl)ethyl)-1H-pyrazole-4-carboxamide; 155N-(2-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethylamino)-2-oxoethyl)-4-methylbenzamide; 156N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethyl)-1-isopropyl-1H-pyrazole-4-carboxamide; 157N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethyl)-2-phenylacetamide; 158 benzyl2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethylcarbamate; 159N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethyl)-4-methylbenzamide; 160N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethyl)-3,4,5-trimethoxybenzamide; 1614-chloro-N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin- D7-yl)ethyl)picolinamide; 1625-bromo-N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin- D7-yl)ethyl)furan-2-carboxamide; 1635-chloro-N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin- D7-yl)ethyl)thiophene-2-carboxamide; 164N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethyl)pyrimidine-4-carboxamide; 165N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethyl)pyrazine-2-carboxamide; 166N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethyl)cyclopentanecarboxamide; 167N-(4H-chromeno[4,3-d]oxazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo- D1,2,3,6-tetrahydropurin-7-yl)acetamide 168N-(3,4-dihydrochromeno[3,4-d]imidazol-2-yl)-2-(1,3-dimethyl- D2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 1692-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(3- Dmethyl-3,4-dihydrochromeno[3,4-d]imidazol-2-yl)acetamide 1702-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(3- Dethyl-3,4-dihydrochromeno[3,4-d]imidazol-2-yl)acetamide 171N-(4H-chromeno[4,3-d]thiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo- D1,2,3,6-tetrahydropurin-7-yl)acetamide 172N-(7-chloro-4H-chromeno[4,3-d]thiazol-2-yl)-2-(1,3-dimethyl- D2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 1732-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(7- Dethoxy-4H-chromeno[4,3-d]thiazol-2-yl)acetamide

TABLE 3 1 2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- A(4-p-tolylthiazol-2-yl)acetamide 22-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- A(4-(4-isopropylphenyl)thiazol-2-yl)acetamide 32-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- A(5-methyl-4-p-tolylthiazol-2-yl)acetamide 4N-(4-(4-tert-butylphenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6- Adioxo-dihydro-2,3-1H-purin-7(6H)-yl)acetamide 5N-(4-(4-cyclohexylphenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6- Adioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 62-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- A(4-(4-ethoxyphenyl)thiazol-2-yl)acetamide 7N-(4-(3,4-dichlorophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6- Adioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 8N-(4-(2,4-difluorophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6- Adioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 92-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- A(4,5-diphenylthiazol-2-yl)acetamide 10N-(4-(4-chlorophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo- A2,3-dihydro-1H-purin-7(6H)-yl)acetamide 11N-(4-(3,4-difluorophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6- Adioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 122-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl)acetamide 132-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(5-phenyl-1,3,4-thiadiazol-2-yl)acetamide 14N-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)-2-(1,3-dimethyl- A2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 15N-(4-(3-bromo-4-methoxyphenyl)thiazol-2-yl)-2-(1,3-dimethyl- D2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 162-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(4-(naphthalen-1-yl)thiazol-2-yl)acetamide 172-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- A(4-(naphthalen-2-yl)thiazol-2-yl)acetamide 18N-(4-(2,4-dichlorophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6- Ddioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 19N-(4,5-dip-tolylthiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Ddihydro-1H-purin-7(6H)-yl)acetamide 20N-(4-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)thiazol-2-yl)-2- D(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)- yl)acetamide 21N-(4-(1H-indol-3-yl)thiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3- Ddihydro-1H-purin-7(6H)-yl)acetamid 222-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- B(4-(5-methylfuran-2-yl)thiazol-2-yl)acetamide 232-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- B(4-(pyridin-2-yl)thiazol-2-yl)acetamide 24N-(4-(4-bromophenyl)-6-(trifluoromethyl)pyrimidin-2-yl)-2- D(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)- yl)acetamide 252-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(4-(2,4-dimethylphenyl)thiazol-2-yl)acetamide 262-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(4-(4-fluorophenyl)thiazol-2-yl)acetamide 27N-(4-(2,5-dimethoxyphenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6- Ddioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 28N-(4-(2,5-difluorophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6- Ddioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 29N-(4-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)thiazol-2-yl)-2-(1,3- Ddimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide 302-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(4-(5-methylthiophen-2-yl)thiazol-2-yl)acetamide 31N-allyl-2-(8-chloro-1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H- Dpurin-7(6H)-yl)-N-(4-phenylthiazol-2-yl)acetamide 322-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N- D(4-(5,6,7,8-tetrahydronaphthalen-2-yl)thiazol-2-yl)acetamide 332-(8-chloro-1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)-N-(4-(adamant-1-yl)phenyl)acetamide; 342-(8-chloro-1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)-N-(4-(4-ethylphenyl)thiazol-2-yl)acetamide; 35N-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7- Dyl)ethyl)-3-(2-methylpyrimidin-4-yl)benzamide; 362-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dethoxyphenyl)-1H-imidazol-2-yl)acetamide 372-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dethoxyphenyl)-5-methyl-1H-imidazol-2-yl)acetamide 382-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dethoxyphenyl)-5-phenyl-1H-imidazol-2-yl)acetamide 392-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dethoxyphenyl)-1-methyl-1H-imidazol-2-yl)acetamide 402-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dethoxyphenyl)-1,5-dimethyl-1H-imidazol-2-yl)acetamide 412-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dethoxyphenyl)-1-methyl-5-phenyl-1H-imidazol-2-yl)acetamide 422-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dethoxyphenyl)-1-ethyl-1H-imidazol-2-yl)acetamide 432-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dethoxyphenyl)-1-ethyl-5-methyl-1H-imidazol-2-yl)acetamide 442-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dethoxyphenyl)-1-ethyl-5-phenyl-1H-imidazol-2-yl)acetamide 452-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dethoxyphenyl)-1-hydroxy-1H-imidazol-2-yl)acetamide 462-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dethoxyphenyl)-1-hydroxy-5-methyl-1H-imidazol-2-yl)acetamide 472-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dethoxyphenyl)-1-hydroxy-5-phenyl-1H-imidazol-2-yl)acetamide 482-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dmethoxyphenyl)thiazol-2-yl)acetamide 492-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Disopropoxyphenyl)thiazol-2-yl)acetamide 502-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- D(dimethylamino)phenyl)thiazol-2-yl)acetamide 51N-(4-(4-(diethylamino)phenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6- Ddioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 52N-(4-(4-acetamidophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6- Ddioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 532-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- Dureidophenyl)thiazol-2-yl)acetamide 54N-(4-(4-cyanophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo- D1,2,3,6-tetrahydropurin-7-yl)acetamide 552-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(4- D(2-hydroxyethoxy)phenyl)thiazol-2-yl)acetamide 56N-(4-(2-chlorophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo- D1,2,3,6-tetrahydropurin-7-yl)acetamide 57N-(4-(3-chlorophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo- D1,2,3,6-tetrahydropurin-7-yl)acetamide 582-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(2- Dethoxyphenyl)thiazol-2-yl)acetamide 592-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(3- Dethoxyphenyl)thiazol-2-yl)acetamide 602-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(2- Dmethoxyphenyl)thiazol-2-yl)acetamide 612-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(3- Dmethoxyphenyl)thiazol-2-yl)acetamide 62N-(4-(2-bromophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo- D1,2,3,6-tetrahydropurin-7-yl)acetamide 63N-(4-(3-bromophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo- D1,2,3,6-tetrahydropurin-7-yl)acetamide 642-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(2- Dfluorophenyl)thiazol-2-yl)acetamide 652-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(3- Dfluorophenyl)thiazol-2-yl)acetamide 662-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-o- Dtolylthiazol-2-yl)acetamide 672-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-m- Dtolylthiazol-2-yl)acetamide 682-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(2- D(2-hydroxyethoxy)phenyl)thiazol-2-yl)acetamide 692-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4-(3- D(2-hydroxyethoxy)phenyl)thiazol-2-yl)acetamide 702-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4- D(3,4,5-trichlorophenyl)thiazol-2-yl)acetamide 712-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4- D(2,3,4-trichlorophenyl)thiazol-2-yl)acetamide 722-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4- D(2,4,5-trichlorophenyl)thiazol-2-yl)acetamide 73N-(4-(3,4-dimethoxyphenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6- Ddioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 74N-(4-(3-chloro-4-ethoxyphenyl)thiazol-2-yl)-2-(1,3-dimethyl- D2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 75N-(4-(4-chloro-3-ethoxyphenyl)thiazol-2-yl)-2-(1,3-dimethyl- D2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 76N-(4-(2-chloro-4-ethoxyphenyl)thiazol-2-yl)-2-(1,3-dimethyl- D2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 77N-(4-(2,4-dichlorophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6- Ddioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 782-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4- D(2,4,6-trichlorophenyl)thiazol-2-yl)acetamide 792-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4- Dphenylthiazol-2-yl)acetamide 802-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4,5- Ddiphenylthiazol-2-yl)acetamide 812-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4,5- Ddi(methylphenyl)thiazol-2-yl)acetamide 82N-(4-(3,4-dichlorophenyl)-5-phenylthiazol-2-yl)-2-(1,3- Ddimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 83N-(4-(3,4-dichlorophenyl)-5-(methylphenyl)thiazol-2-yl)-2-(1,3- Ddimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 84N-(4-(3,4-dichlorophenyl)-5-(4-ethoxyphenyl)thiazol-2-yl)-2- D(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 85N-(4,5-bis(3,4-dichlorophenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6- Ddioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 86N-(4-(4-ethoxyphenyl)-5-phenylthiazol-2-yl)-2-(1,3-dimethyl- D2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 87N-(4-(4-ethoxyphenyl)-5-(methylphenyl)thiazol-2-yl)-2-(1,3- Ddimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 88N-(4-(4-ethoxyphenyl)-5-(4-ethoxyphenyl)thiazol-2-yl)-2-(1,3- Ddimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 89N-(5-(3,4-dichlorophenyl)-4-(4-ethoxyphenyl)thiazol-2-yl)-2- D(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 902-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(5- Dphenylthiazol-2-yl)acetamide 912-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(5- D(methylphenyl)thiazol-2-yl)acetamide 922-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(5- D(chlorophenyl)thiazol-2-yl)acetamide 932-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(5- D(methoxyphenyl)thiazol-2-yl)acetamide 942-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(5- D(ethoxyphenyl)thiazol-2-yl)acetamide 952-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4- D(pyridin-4-yl)thiazol-2-yl)acetamide 962-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4- D(pyridin-3-yl)thiazol-2-yl)acetamide 972-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4- D(pyrimidin-2-yl)thiazol-2-yl)acetamide 982-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4- Dphenyloxazol-2-yl)acetamide 992-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(5- Dethyl-4-phenyloxazol-2-yl)acetamide 1002-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(4,5- Ddiphenyloxazol-2-yl)acetamide 101N-(4,5-dip-tolyloxazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo-1,2,3,6- Dtetrahydropurin-7-yl)acetamide 102N-(4,5-bis(4-methoxyphenyl)oxazol-2-yl)-2-(1,3-dimethyl-2,6- Ddioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 103N-(4-(3,4-dichlorophenyl)-5-fluorothiazol-2-yl)-2-(1,3-dimethyl- D2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 104N-(5-cyano-4-(3,4-dichlorophenyl)thiazol-2-yl)-2-(1,3-dimethyl- D2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 105N-(4-(3,4-dichlorophenyl)-5-methoxythiazol-2-yl)-2-(1,3- Ddimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 106N-(4-(3,4-dichlorophenyl)-5-methylthiazol-2-yl)-2-(1,3- Ddimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 107N-(4-(3,4-dichlorophenyl)-5-ethylthiazol-2-yl)-2-(1,3-dimethyl- D2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 108N-(4-(3,4-dichlorophenyl)-5-(trifluoromethyl)thiazol-2-yl)-2- D(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 1092-(4-(3,4-dichlorophenyl)-2-(2-(1,3-dimethyl-2,6-dioxo-1,2,3,6- Dtetrahydropurin-7-yl)acetamido)thiazol-5-yl)ethyl dihydrogen phosphate110 N-(4-(3,4-dichlorophenyl)-5-methylthiazol-2-yl)-2-(1,3- Ddimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 111N-(4-(3,4-dichlorophenyl)-5-ethylthiazol-2-yl)-2-(1,3-dimethyl- D2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 112N-(4-(3,4-dichlorophenyl)-5-isopropylthiazol-2-yl)-2-(1,3- Ddimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide 113N-(4-(3,4-dichlorophenyl)-5-(2-hydroxyethyl)thiazol-2-yl)-2- D(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)acetamide

Additional exemplary compounds are provided in Table 4, FIG. 6.

One aspect of the present invention provides a pharmaceuticalpreparation suitable for use in a human patient, or for veterinary use,comprising an effective amount of any of the compounds shown above(e.g., a compound described hereing or a salt thereof, or a solvate,hydrate, oxidative metabolite or prodrug of the compound or its salt),and one or more pharmaceutically acceptable excipients. In certainembodiments, the pharmaceutical preparations may be for use in treatingor preventing a condition involving activation of TRPA1 or for whichreduced TRPA1 activity can reduce the severity. In certain embodiments,the pharmaceutical preparations have a low enough pyrogen activity to besuitable for use in a human patient, or for veterinary use. In certainembodiments, the pharmaceutical preparation comprises an effectiveamount of any of the compounds shown above, wherein the compoundinhibits TRPA1 with an IC₅₀ of 10 micromolar or less. In certainembodiments, the pharmaceutical preparation comprises a compound whichinhibits TRPA1 with an IC₅₀ of 1 micromolar or less, or even with anIC₅₀ of 500 nM or less, 250 nM or less, 200 nM or less, or even 100 nMor less.

In certain embodiments, the TRPA1 inhibitor for use in methods orpharmaceutical preparations of the present invention is selected from acompound depicted in Tables 1, 3, or 4. In certain embodiments, thepresent invention contemplates the use of any compound as depicted inoptionally substituted in any of the methods or pharmaceuticalpreparations of the present invention.

One aspect of the current invention provides use of a TRPA1 inhibitor inthe manufacture of a medicament for treating or preventing a conditioninvolving activation of TRPA1 or for which reduced TRPA1 activity canreduce the severity, wherein the TRPA1 inhibitor is represented by anyof the compounds shown above (e.g., a compound described herein or asalt thereof, or a solvate, hydrate, oxidative metabolite or prodrug ofthe compound or its salt). In certain embodiments, the compound inhibitsa TRPA1 mediated current with an IC₅₀ of less than 10 micromolar.

In certain embodiments of the above formula, substituted substituentsmay be substituted with one or more of: alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl,heterocyclylalkyl, aralkyl, or heteroaralkyl, any of which may itself befurther substituted, or halogen, hydroxyl, carbonyl (e.g., ester,carboxyl, or formyl), thiocarbonyl (e.g., thioester, thiocarboxylate, orthioformate), ketone, aldehyde, amino, acylamino, amido, amidino, cyano,nitro, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, and phosphoryl.

In certain embodiments, the invention contemplates that any of theparticular compounds depicted in Tables 1, 3, or 4 can be administeredto treat any of the diseases or conditions disclosed herein. In someembodiments, the compound is formulated as a pharmaceutical preparationprior to administration. In certain embodiments, the TRPA1 inhibitor foruse in methods or pharmaceutical preparations of the present inventionis selected from a compound depicted in Tables 1, 3, or 4. In certainembodiments, the present invention contemplates the use of any compoundas depicted in Tables 1, 3, or 4 in any of the methods or pharmaceuticalpreparations of the present invention.

The particular compounds and structural formulas disclosed herein aremerely exemplary. The use of small molecule TRPA1 inhibitors having oneor more of the functional or structural characteristics described hereinare similarly contemplated,

Compounds of any of the above structures may be used in the manufactureof medicaments for the treatment of any diseases disclosed herein.

Compounds of any of the above structures may be used to inhibit afunction of a TRPA1 channel in vitro or in vivo.

In certain embodiments, compounds that include all or a functionalportion of any of the foregoing structures may be used in themanufacture of medicaments for the treatment of any of the diseasesdisclosed herein. Additionally or alternatively, such compounds may beused in in vitro or in vivo methods of inhibiting TRPA1 function, suchas a TRPA1-mediated current.

In certain embodiments, the TRPA1 antagonist for use in the methods ofthe present invention is a small molecule that is not an aminoglycoside.

In particular embodiments, a small molecule TRPA1 antagonist is chosenfor use because it is more selective for one TRP isoform than others,e.g., 10-fold, and more preferably at least 20, 40, 50, 60, 70, 80, orat least 100- or even 1000-fold more selective for TRPA1 over one ormore of TRPC6, TRPV5, TRPV6, TRPM8, TRPV1, TRPV2, TRPV4, and/or TRPV3.In other embodiments, the differential is smaller, e.g., it morestrongly inhibits TRPA1 than TRPM8, TRPV1, TRPV2, TRPV3, and/or TRPV4,preferably at least twice, three times, five times, or even ten timesmore strongly. Such comparisons may be made, for example, by comparingIC₅₀ values.

In particular embodiments, a small molecule TRPA1 antagonist is chosenfor use because it is more selective for one TRPA1 than for othernon-TRP ion channels, e.g., 10-fold, and more preferably at least 20,40, 50, 60, 70, 80, or at least 100- or even 1000-fold more selectivefor TRPA1 over one or more of NaV1.2, Cav1.2, Cav3.1, HERG, and/ormitochondrial uniporter. In other embodiments, the differential issmaller, e.g., it more strongly inhibits TRPA1 than NaV1.2, Cav1.2,Cav3.1, HERG, and/or mitochondrial uniporter, preferably at least twice,three times, five times, or even ten times more strongly. Suchcomparisons may be made, for example, by comparing IC₅₀ values.

In certain embodiments, a compound which is an antagonist of TRPA1 ischosen to selectively antagonize TRPA1 over other ion channels, e.g.,the compound modulates the activity of TRPA1 at least an order ofmagnitude more strongly than it modulates the activity of one or more ofNaV1.2, Cav1.2, Cav3.1, HERG, and/or mitochondrial uniporter, preferablyat least two orders of magnitude more strongly, even more preferably atleast three orders of magnitude more strongly. In certain embodiments,the compound modulates the activity of TRPA1 at least 1.5 orders ofmagnitude more strongly than the activity of one or more of NaV1.2,Cav1.2, Cav3.1, HERG, or mitochondrial uniporter. Such comparisons maybe made, for example, by comparing IC₅₀ values.

Similarly, in particular embodiments, a small molecule is chosen for usebecause it lacks significant activity against one or more targets otherthan TRPA1. For example, the compound may have an IC₅₀ above 500 nM,above 1 μM, or even above 10 μM or 100 μM for inhibiting one or more ofTRPC6, TRPV5, TRPV6, Cav1.2, Cav3.1, NaV1.2, HERG, and the mitochondrialuniporter.

In particular embodiments, the small molecule is chosen for use becauseit is more selective for one TRP isoform than others, e.g., 10-fold, andmore preferably at least 100- or even 1000-fold more selective for TRPA1over one or more of TRPC6, TRPV5, TRPV6, TRPM8, TRPV1, HERG, NaV1.2,mitochondrial uniporter, TRPV3 and/or TRPV4. In other embodiments, thedifferential is smaller, e.g., it more strongly inhibits TRPA1 thanTRPM8, TRPV1 and/or TRPV4, preferably at least twice, three times, fivetimes, or even ten times more strongly. Such comparisons may be made,for example, by comparing IC₅₀ values.

In certain embodiment, a small molecule is chosen because it antagonizesthe function of both TRPA1 and TRPM8, TRPV1 and/or TRPV3. Although suchcompounds selectively antagonize the function of both ion channels, theIC₅₀ values need not be identical.

In certain embodiments of any of the foregoing, the small molecule maybe chosen because it is capable of inhibiting receptor-mediated (orcold/stress mediated) activation of TRPA1. In certain embodiments, theTRPA1 antagonist inhibits receptor mediated activation of TRPA1 andmustard oil induced activation of TRPA1. In certain other embodiments,the TRPA1 antagonist inhibits receptor operated activation of TRPA1 butdoes not inhibit mustard oil induced activation of TRPA1. In certainother embodiments, the TRPA1 antagonist inhibits mustard oil inducedactivation of TRPA1 but does not inhibit cold mediated activation ofTRPA1.

In certain embodiments of any of the foregoing, the small molecule maybe chosen because it inhibits a TRPA1 function with an IC₅₀ less than orequal to 1 uM, or even less than or equal to 700, 600, 500, 400, 300,250, 200, or 100 nM. In other embodiments, the small molecule is chosenbecause it inhibits a TRPA1 function with an IC₅₀ less than or equal to75 nM, less than or equal to 50 nM, or even less than or equal to 25,10, 5, or 1 nM. In certain other embodiments of any of the foregoing,the small molecule inhibits TRPA1 function with an IC₅₀ less than orequal to 10 micromolar or less than or equal to 5 micromolar or lessthan or equal to 2.5 micromolar or less than or equal to 1.5 micromolar.

In certain embodiments of any of the foregoing, the compound may bechosen based on the rate of inhibition of a TRPA1 function. In oneembodiment, the compound inhibits a TRPA1 function in less than 5minutes, preferably less than 4, 3, or 2 minutes. In another embodiment,the compound inhibits a TRPA1 function in less than about 1 minute. Inyet another embodiment, the compound inhibits a TRPA1 function in lessthan about 30 seconds.

In any of the foregoing embodiments, the small molecule antagonist ofTRPA1 function may inhibit the outward current, the inward current, orany combination of one or more of these currents. Compounds that inhibitmore than one of the foregoing currents may do so with the same or withdiffering IC₅₀ values. In any of the foregoing, the ability of acompound to inhibit a particular current can be assessed either in vitroor in vivo. Compounds that inhibit any of the foregoing currents in anin vitro or in vivo assay are characterized as compounds that inhibit afunction of TRPA1. Stated another way, an exemplary function of TRPA1that may be inhibited by the present compounds is a TRPA1-mediatedcurrent. Additionally or alternatively, a further exemplary function ofTRPA1 that may be inhibited by the present compounds is ion fluxmediated by TRPA1.

In any of the foregoing or following embodiments, the small molecule ischaracterized by some level of activity versus other ion channels (e.g.,certain compounds are selective for inhibiting TRPA1 and other compoundsexhibit a level of cross reactivity against one or more other ionchannel). When a small molecule is characterized by its activity againstanother ion channel, inhibition of a function or activity of the otherion channel is defined analogously to the way in which a function of aTRPA1 channel is defined. Thus, inhibiting the function of another ionchannel means, for example, inhibiting ion flux mediated by that otherion channel or inhibiting the current mediated by that other ionchannel.

In certain embodiments of any of the foregoing, inhibition of a TRPA1function means that a function, for example a TRPA1 mediated current, isdecreased by greater than 50% in the presence of an effective amount ofa compound in comparison to in the absence of the compound or incomparison to an ineffective amount of a compound. In certain otherembodiments, the inhibition of a TRPA1 function means that a function,for example a TRPA1 mediated current or TRPA1 mediated ion flux, isdecreased by at least 50%, 60%, 70%, 75%, 80%, 85%, or 90% in thepresence of an effective amount of a compound in comparison to in theabsence of the compound. In still other embodiments, the inhibition of aTRPA1 function means that a function, for example a TRPA1 mediatedcurrent, is decreased by at least 92%, 95%, 97%, 98%, 99%, or 100% inthe presence of an effective amount of a compound in comparison to inthe absence of the compound.

In any of the foregoing embodiments, IC₅₀ values are measured in vitrousing, for example, patch clamp analysis or standard measurements ofcalcium flux. Exemplary in vitro methods for calcium flux-based IC₅₀estimation are described in Example 1. Methods used to obtain moredefinitive IC₅₀ measurements are described in Example 2. Alternatively,estimates of % inhibition of current or ion flux can also be calculatedand used to assess efficacy of a compound as an inhibitor.

Without being bound by theory, a compound may inhibit a function ofTRPA1 by binding covalently or non-covalently to a portion of TRPA1.Alternatively, a compound may inhibit a function of TRPA1 indirectly,for example, by associating with a protein or non-protein cofactornecessary for a function of TRPA1. One of skill in the art will readilyappreciate that an inhibitory compound may associate reversibly orirreversibly with TRPA1 or a cofactor thereof. Compounds that reversiblyassociate with TRPA1 or a cofactor thereof may continue to inhibit afunction of TRPA1 even after dissociation.

In certain embodiments of any of the foregoing, the compound thatinhibits a function of TRPA1 is a small organic molecule or a smallinorganic molecule. Exemplary small molecules include, but are notlimited to, small molecules that bind to a TRPA1 channel and inhibit oneor more function of a TRPA1 channel.

In certain embodiments of any of the foregoing, the TRPA1 inhibitor isused to treat or ameliorate pain. Exemplary classes of pain that cantreated using a TRPA1 inhibitor include, but are not limited tonociceptive pain, inflammatory pain, and neuropathic pain. Pain that canbe treated with a TRPA1 inhibitor can be chronic or acute. Throughoutthe specification, a variety of conditions and diseases characterized,at least in part, by pain are discussed in detail. The inventioncontemplates that the pain associated with any of these diseases orconditions can be treated using any of the TRPA1 inhibitors describedherein. The inhibitor can be formulated in a pharmaceutical preparationappropriate for the intended route of administration.

In certain embodiments, the TRPA1 inhibitor is non-narcotic and haslittle or no narcotic side-effects. In certain other embodiments, theTRPA1 inhibitor can be used to treat or ameliorate pain with fewerside-effects than narcotic pain relievers. Exemplary side-effects thatmay be substantially absent at effective dosages of TRPA1 inhibitorsinclude one or more of exopthalmos, catalepsy, disruption of gutmotility, and inhibition of sensation in non-injured areas of the body.

In certain embodiments, the TRPA1 inhibitor can be used to treatincontinence. In certain embodiments, the TRPA1 inhibitor is used toreduce bladder hyperactivity by decreasing the activity of the neuronsthat innervate the bladder. In certain embodiments, incontinence isaccompanied by pain. For example, incontinence incident to bladdercystitis or incontinence incident to an injury may be accompanied bypain. When incontinence is accompanied by pain, a TRPA1 inhibitor may beadministered to treat both incontinence and to reduce pain.

The subject TRPA1 inhibitors can be used alone or in combination withother pharmaceutically active agents. Examples of such otherpharmaceutically active agents include, but are not limited to,anti-inflammatory agents (e.g., NSAIDS, bradykinin receptor antagonists,hormones and autacoids such as corticosteroids), anti-acne agents (e.g.,retinoids), anti-wrinkle agents, anti-scarring agents, anti-incontinenceagents (such as M1-receptor antagonists) anti-emetics (such as NK1antagonists), anti-psoriatic agents, antacids, anti-proliferative agents(e.g., anti-eczema agents, anti-cancer), anti-fungal agents, anti-viralagents, anti-septic agents (e.g., antibacterials), local anaesthetics,anti-migraine agents, keratolytic agents, hair growth stimulants, hairgrowth inhibitors, and other agents used for the treatment of skindiseases or conditions. Certain active agents belong to more than onecategory.

For any of the foregoing, a TRPA1 inhibitor can be formulated foradministration by a route appropriate for the disease or injury beingtreated. For example, the TRPA1 inhibitor can be formulated, forexample, for oral, transdermal, topical, intraperitoneal, intravenous,intravascular, intrathecal, intrapericardial, intramyocardial,subcutaneous, rectal, vaginal, or urethral delivery. Furthermore, theTRPA1 inhibitor can be formulated for delivery via a device. Exemplarydevices include, but are not limited to, a catheter, wire, stent, orother intraluminal device. Further exemplary delivery devices alsoinclude a patch, bandage, mouthguard, or dental apparatus.

The invention contemplates pharmaceutical compositions of any of theforegoing TRPA1 inhibitors. Exemplary pharmaceutical compositions areformulated in a pharmaceutically acceptable carrier.

The subject TRPA1 inhibitors can be used alone or as part of atherapeutic regimen combined with other treatments, therapies, orinterventions appropriate for the particular disease, condition, injuryor disorder being treated. When used as part of a therapeutic regimen,the invention contemplates use of TRPA1 inhibitors in combination withone or more of the following treatment modalities: administration ofnon-TRPA1 inhibitor pharmaceuticals, chemotherapy, radiotherapy,homeopathic therapy, diet, stress management, and surgery.

When administered alone or as part of a therapeutic regimen, in certainembodiments, the invention contemplates administration of TRPA1inhibitors to treat a particular primary disease, injury, disorder, orcondition. Additionally or alternatively, the invention contemplatesadministration of TRPA1 inhibitors to treat pain associated with adisease, injury, disorder, or condition. In still other embodiments, theinvention contemplates administration of TRPA1 inhibitors to treatsymptoms secondary to the primary disease, injury, disorder, orconditions.

The invention contemplates pharmaceutical preparations and uses of TRPA1antagonists having any combination of the foregoing or followingcharacteristics, as well as any combination of the structural orfunctional characteristics of the TRPA1 antagonists described herein.Any such antagonists or preparations can be used in the treatment of anyof the diseases or conditions described herein. Additionally, theinvention contemplates the use of any such antagonists or preparationsfor inhibiting a TRPA1 mediated current in vitro. Combinations of any ofthe foregoing or following aspects and embodiments of the invention arealso contemplated. For example, the invention contemplates that TRPA1antagonists having any of the particular potencies and specificitiesoutlined herein can be formulated for the appropriate route ofadministration and can be used in treating any of the conditions ordiseases detailed herein.

In certain embodiments of any of the foregoing, TRPA1 antagonistcompounds for use in the methods of the present invention have one ormore of any of the foregoing properties (e.g., IC₅₀, specificity,selectivity, activity, formulation, etc.). Compounds and uses ofantagonist compounds having any combination of the foregoing propertiesare specifically contemplated.

The terms “antagonist” and “inhibitor” are used interchangeably to referto an agent that decreases or suppresses a biological activity, such asto repress an activity of an ion channel, such as TRPA1. TRPA1inhibitors include inhibitors having any combination of the structuraland/or functional properties disclosed herein.

An “effective amount” of, e.g., a TRPA1 antagonist, with respect to thesubject methods of inhibition or treatment, refers to an amount of theantagonist in a preparation which, when applied as part of a desireddosage regimen brings about a desired clinical or functional result.Without being bound by theory, an effective amount of a TRPA1 antagonistfor use in the methods of the present invention, includes an amount of aTRPA1 antagonist effective to decrease one or more in vitro or in vivofunction of a TRPA1 channel. Exemplary functions include, but are notlimited to, membrane polarization (e.g., an antagonist may promotehyperpolarization of a cell), ion flux, ion concentration in a cell,outward current, and inward current. Compounds that antagonize TRPA1function include compounds that antagonize an in vitro or in vivofunctional activity of TRPA1. When a particular functional activity isonly readily observable in an in vitro assay, the ability of a compoundto inhibit TRPA1 function in that in vitro assay serves as a reasonableproxy for the activity of that compound. In certain embodiments, aneffective amount is an amount sufficient to inhibit a TRPA1-mediatedcurrent and/or the amount sufficient to inhibit TRPA1 mediated ion flux.

The TRPA1 inhibitors for use in the methods of the present invention maybe characterized according to their activity, or lack of activity,against one or more other ion channels. When other ion channels arereferred to, inhibition of a function of such other ion channels isdefined similarly. For example, inhibition of an ion channel or anactivity of an ion channel means the antagonist inhibits one or morefunctional activities of the other ion channel. Such functions includethe current mediated by the particular ion channel, ion flux, ormembrane polarization.

The term “nucleic acid” refers to a polymeric form of nucleotides,either ribonucleotides or deoxynucleotides or a modified form of eithertype of nucleotide. The terms should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs,and, as applicable to the embodiment being described, single-stranded(such as sense or antisense) and double-stranded polynucleotides.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “polypeptide”, and the terms “protein” and “peptide” which areused interchangeably herein, refers to a polymer of amino acids.Exemplary polypeptides include gene products, naturally-occurringproteins, homologs, orthologs, paralogs, fragments, and otherequivalents, variants and analogs of the foregoing.

The term “prodrug” is intended to encompass compounds that, underphysiological conditions, are converted into the therapeutically activeagents of the present invention. A common method for making a prodrug isto include selected moieties that are hydrolyzed under physiologicalconditions to reveal the desired molecule. In other embodiments, theprodrug is converted by an enzymatic activity of the host animal.

The term “sequence identity” means that sequences are identical (i.e.,on a nucleotide-by-nucleotide basis for nucleic acids or aminoacid-by-amino acid basis for polypeptides) over a window of comparison.The term “percentage of sequence identity” is calculated by comparingtwo optimally aligned sequences over the comparison window, determiningthe number of positions at which the identical amino acids occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thecomparison window, and multiplying the result by 100 to yield thepercentage of sequence identity. Methods to calculate sequence identityare known to those of skill in the art and described in further detailbelow.

The term “small molecule” refers to a compound having a molecular weightless than about 2500 amu, preferably less than about 2000 amu, even morepreferably less than about 1500 amu, still more preferably less thanabout 1000 amu, or most preferably less than about 750 amu.

The terms “stringent conditions” or “stringent hybridization conditions”refer to conditions which promote specific hybridization between twocomplementary polynucleotide strands so as to form a duplex. Stringentconditions may be selected to be about 5° C. lower than the thermalmelting point (Tm) for a given polynucleotide duplex at a defined ionicstrength and pH. The length of the complementary polynucleotide strandsand their GC content will determine the Tm of the duplex, and thus thehybridization conditions necessary for obtaining a desired specificityof hybridization. The Tm is the temperature (under defined ionicstrength and pH) at which 50% of the a polynucleotide sequencehybridizes to a perfectly matched complementary strand. In certain casesit may be desirable to increase the stringency of the hybridizationconditions to be about equal to the Tm for a particular duplex. Incertain embodiments, stringent hybridization conditions include a washstep of 0.2×SSC at 65° C.

The terms “TRPA1”, “TRPA1 protein”, and “TRPA1 channel” are usedinterchangeably throughout the application. These terms refer to an ionchannel (e.g., a polypeptide) comprising the amino acid sequence setforth in SEQ ID NO: 1, SEQ ID NO:3, or SEQ ID NO: 5, or an equivalentpolypeptide, or a functional bioactive fragment thereof. In certainembodiments, the term refers to a polypeptide comprising, consisting of,or consisting essentially of, the amino acid sequence set forth in SEQID NO: 1, SEQ ID NO:3, or SEQ ID NO: 5. TRPA1 includes polypeptides thatretain a function of TRPA1 and comprise (i) all or a portion of theamino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5; (ii) the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:3or SEQ ID NO: 5 with 1 to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 ormore conservative amino acid substitutions; (iii) an amino acid sequencethat is at least 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO: 5; and (iv)functional fragments thereof. Polypeptides of the invention also includehomologs, e.g., orthologs and paralogs, of SEQ ID NO: 1, SEQ ID NO: 3 orSEQ ID NO: 5.

The term “TRPA1” further refers to a nucleic acid encoding a polypeptideof the invention, e.g., a nucleic acid comprising a sequence consistingof, or consisting essentially of, the polynucleotide sequence set forthin SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. A nucleic acid of theinvention may comprise all, or a portion of: the nucleotide sequence ofSEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; a nucleotide sequence atleast 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ IDNO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; a nucleotide sequence thathybridizes under stringent conditions to SEQ ID NO: 2, SEQ ID NO: 4 orSEQ ID NO: 6; nucleotide sequences encoding polypeptides that arefunctionally equivalent to polypeptides of the invention; nucleotidesequences encoding polypeptides at least about 70%, 75%, 80%, 85%, 90%,95%, 98%, 99% homologous or identical with an amino acid sequence of SEQID NO: 1, SEQ ID NO:3 or SEQ ID NO: 5; nucleotide sequences encodingpolypeptides having an activity of a polypeptide of the invention andhaving at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or morehomology or identity with SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO: 5;nucleotide sequences that differ by 1 to about 2, 3, 5, 7, 10, 15, 20,30, 50, 75 or more nucleotide substitutions, additions or deletions,such as allelic variants, of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6;nucleic acids derived from and evolutionarily related to SEQ ID NO: 2,SEQ ID NO: 4 or SEQ ID NO: 6; and complements of, and nucleotidesequences resulting from the degeneracy of the genetic code, for all ofthe foregoing and other nucleic acids of the invention. Nucleic acids ofthe invention also include homologs, e.g., orthologs and paralogs, ofSEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 and also variants of SEQ IDNO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 which have been codon optimized forexpression in a particular organism (e.g., host cell). Where notexplicitly stated, one of skill in the art can readily assess whetherTRPA1 refers to a nucleic acid or a protein.

The term “oxidative metabolite” is intended to encompass compounds thatare produced by metabolism of the parent compound under normalphysiological conditions. Specifically, an oxidative metabolite isformed by oxidation of the parent compound during metabolism. Forexample, a thioether group may be oxidized to the correspondingsulfoxide or sulfone.

The term “solvate” as used herein, refers to a compound formed bysolvation (e.g., a compound formed by the combination of solventmolecules with molecules or ions of the solute).

The term “hydrate” as used herein, refers to a compound formed by theunion of water with the parent compound.

The term “treating” includes prophylactic and/or therapeutic treatments.The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The terms “compound” and “agent” are used interchangeably to refer tothe inhibitors/antagonists of the invention. In certain embodiments, thecompounds are small organic or inorganic molecules, e.g., with molecularweights less than 7500 amu, preferably less than 5000 amu, and even morepreferably less than 2000, 1500, 1000, or 500 amu. One class of smallorganic or inorganic molecules are non-peptidyl, e.g., containing 2, 1,or no peptide and/or saccharide linkages. In certain other embodiments,the compounds are peptidyl agents such as polypeptides or antibodies. Incertain other embodiments, the compounds are proteins, for example,antibodies or aptamers. Such compounds can bind to and inhibit afunction of TRPA1. In certain other embodiments, the compounds arenucleic acids, for example, TRPA1 antisense oligonucleotides or TRPA1RNAi constructs. Such compounds can inhibit the expression of TRPA1,thereby inhibiting the activity of TRPA1. Other exemplary compounds thatmay act as inhibitors include ribozymes and peptide fragments.

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbylC(O)—, preferably alkylC(O)—.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′11 represents a hydrogen, analkyl, an alkenyl or —(CH2)m-R8, where m and R8 are as defined above.

Herein, the term “aliphatic group” refers to a straight-chain,branched-chain, or cyclic aliphatic hydrocarbon group and includessaturated and unsaturated aliphatic groups, such as an alkyl group, analkenyl group, and an alkynyl group.

The term “alkenyl”, as used herein, refers to an aliphatic groupcontaining at least one double bond and is intended to include both“unsubstituted alkenyls” and “substituted alkenyls”, the latter of whichrefers to alkenyl moieties having substituents replacing a hydrogen onone or more carbons of the alkenyl group. Such substituents may occur onone or more carbons that are included or not included in one or moredouble bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed below, except where stability isprohibitive. For example, substitution of alkenyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined below, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—-(CH2)m-R8,where m and R8 are described above.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C1-C30 for straight chains, C3-C30 for branchedchains), and more preferably 20 or fewer, and most preferably 10 orfewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms intheir ring structure, and more preferably have 5, 6 or 7 carbons in thering structure.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, anamido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl,an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

The term “alkynyl”, as used herein, refers to an aliphatic groupcontaining at least one triple bond and is intended to include both“unsubstituted alkynyls” and “substituted alkynyls”, the latter of whichrefers to alkynyl moieties having substituents replacing a hydrogen onone or more carbons of the alkynyl group. Such substituents may occur onone or more carbons that are included or not included in one or moretriple bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed above, except where stability isprohibitive. For example, substitution of alkynyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethylthio, and thelike.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈, or R₉ and R₁₀ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In preferred embodiments, only one of R₉ or R₁₀can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not forman imide. In certain such embodiments, neither R₉ and R₁₀ is attached toN by a carbonyl, e.g., the amine is not an amide or imide, and the amineis preferably basic, e.g., its conjugate acid has a pK_(a) above 7. Ineven more preferred embodiments, R₉ and R₁₀ (and optionally R′₁₀) eachindependently represent a hydrogen, an alkyl, an alkenyl, or—(CH₂)_(m)—R₈. Thus, the term “alkylamine” as used herein means an aminegroup, as defined above, having a substituted or unsubstituted alkylattached thereto, i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides that may be unstable.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,polycyclyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl,aromatic or heteroaromatic moieties, —CF3, —CN, or the like. The term“aryl” also includes polycyclic ring systems having two or more cyclicrings in which two or more carbons are common to two adjoining rings(the rings are “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The term “carbocycle or cyclyl”, as used herein, refers to an aromaticor non-aromatic ring in which each atom of the ring is carbon.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The term “electron withdrawing group” refers to chemical groups whichwithdraw electron density from the atom or group of atoms to whichelectron withdrawing group is attached. The withdrawal of electrondensity includes withdrawal both by inductive and bydelocalization/resonance effects. Examples of electron withdrawinggroups attached to aromatic rings include perhaloalkyl groups, such astrifluoromethyl, halogens, azides, carbonyl containing groups such asacyl groups, cyano groups, and imine containing groups.

The term “ester”, as used herein, refers to a group —C(O)OR⁹ wherein R⁹represents a hydrocarbyl group.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a hetaryl group.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively). Any ring atom can besubstituted (e.g., by one or more substituents).

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, anaromatic or heteroaromatic moiety, —CF₃, —CN, or the like.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heteroaryl” and “hetaryl” also include polycyclic ring systems havingtwo or more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heterocyclyl” and “heterocyclic” also include polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to behydrocarbyl for the purposes of this application, but substituents suchas acetyl (which has a ═O substituent on the linking carbon) and ethoxy(which is linked through oxygen, not carbon) are not. Hydrocarbyl groupsinclude, but are not limited to aryl, heteroaryl, carbocycle,heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are ten or fewer atoms in the substituent,preferably six or fewer. A “lower alkyl”, for example, refers to analkyl group that contains ten or fewer carbon atoms, preferably six orfewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl,or alkoxy substituents defined herein are respectively lower acyl, loweracyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy,whether they appear alone or in combination with other substituents,such as in the recitations hydroxyalkyl and aralkyl (in which case, forexample, the atoms within the aryl group are not counted when countingthe carbon atoms in the alkyl substituent).

As used herein, the term “nitro” means —NO2; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO2-.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF3, —CN, or the like.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R9 and R10 are as defined above.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R41 is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that canbe represented by the general formula:

in which R9 and R′11 are as defined above.

The term “sulfonate” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R41 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the general formula:

in which R44 is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

The term “thioester”, as used herein, refers to a group —C(O)SR⁹ or—SC(O)R⁹ wherein R⁹ represents a hydrocarbyl.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (d)-isomers, (l)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Methods of preparing substantially isomerically pure compounds are knownin the art. If, for instance, a particular enantiomer of a compound ofthe present invention is desired, it may be prepared by asymmetricsynthesis, or by derivation with a chiral auxiliary, where the resultingdiastereomeric mixture is separated and the auxiliary group cleaved toprovide the pure desired enantiomers. Alternatively, where the moleculecontains a basic functional group, such as amino, or an acidicfunctional group, such as carboxyl, diastereomeric salts may be formedwith an appropriate optically active acid or base, followed byresolution of the diastereomers thus formed by fractionalcrystallization or chromatographic means well known in the art, andsubsequent recovery of the pure enantiomers. Alternatively,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Techniques for inverting or leaving unchanged a particular stereocenter,and those for resolving mixtures of stereoisomers are well known in theart, and it is well within the ability of one of skill in the art tochoose an appropriate method for a particular situation. See, generally,Furniss et al. (eds.), Vogel's Encyclopedia of Practical OrganicChemistry 5^(th) Ed., Longman Scientific and Technical Ltd., Essex,1991, pp. 809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., the ability to inhibit TRPA1activity), wherein one or more simple variations of substituents aremade which do not adversely affect the efficacy of the compound. Ingeneral, the compounds of the present invention may be prepared by themethods illustrated in the general reaction schemes as, for example,described below, or by modifications thereof, using readily availablestarting materials, reagents and conventional synthesis procedures. Inthese reactions, it is also possible to make use of variants which arein themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²¹I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

The symbol

, whether utilized as a bond or displayed perpendicular to a bondindicates the point at which the displayed moiety is attached to theremainder of the molecule, solid support, etc.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

The term “pharmaceutically acceptable salts” includes salts of theactive compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituents found on the compoundsdescribed herein. When compounds of the present invention containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the present invention containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable acid addition salts includethose derived from inorganic acids like hydrochloric, hydrobromic,nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, trifluoroacetic, propionic, isobutyric, maleic, malonic,benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic,benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, andthe like. Also included are the salts of amino acids such as arginateand the like, and salts of organic acids like glucuronic or galactunoricacids and the like (see, for example, Berge et al., “PharmaceuticalSalts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certainspecific compounds of the present invention contain both basic andacidic functionalities that allow the compounds to be converted intoeither base or acid addition salts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers form the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentinvention.

The term “low enough pyrogen activity”, with reference to apharmaceutical preparation, refers to a preparation that does notcontain a pyrogen in an amount that would lead to an adverse effect(e.g., irritation, fever, inflammation, diarrhea, respiratory distress,endotoxic shock, etc.) in a subject to which the preparation has beenadministered. For example, the term is meant to encompass preparationsthat are free of, or substantially free of, an endotoxin such as, forexample, a lipopolysaccharide (LPS).

Diseases, Disorders, or Conditions Related to TRPA1 Function

In certain embodiments, the invention provides methods and compositionsfor inhibiting a function of a TRPA1 channel in vitro or in vivo.Exemplary functions include, but are not limited to, TRPA1-mediatedcurrent. In certain embodiments, the invention provides methods forpreventing or treating a disease or disorder or condition byadministering an agent that modulates the level and/or activity of aTRPA1 protein. In other embodiments, the compound selectively inhibitsthe expression level and/or activity of a TRPA1 protein. In other words,in certain embodiment, the compound inhibits the activity of a TRPA1protein preferentially in comparison to the activity of one or moreother ion channels.

In particular embodiments of the methods for preventing or treatingdiseases and disorders provided herein, the disease or disorder can be,for example, a pain or sensitivity to touch such as pain related to adisease or disorder, e.g., cancer pain, a dermatological disease ordisorder, e.g., psoriasis and basal cell and squamous cell cariconomas,a neurodegenerative disease or disorder, e.g., Alzheimer's disease (AD),Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis(ALS), and other brain disorders caused by trauma or other insultsincluding aging, an inflammatory disease (e.g., asthma, chronicobstructive pulmonary disease, rheumatoid arthritis, osteoarthritis,inflammatory bowel disease, glomerulonephritis, neuroinflammatorydiseases, multiple sclerosis, and disorders of the immune system),cancer (e.g. liposarcoma) or other proliferative disease, kidney diseaseand liver disease, a metabolic disorder such as diabetes. Furtherdiseases and conditions include post-surgical pain, post herpeticneuralgia, incontinence, and shingles.

Because of the important role that calcium regulation plays in manycellular processes including cellular activation, gene expression,cellular trafficking and apoptotic cell death, calcium dyshomeostasis isimplicated in the many diseases and disorders involving such cellularactivities. These diseases and disorders include dermatological diseasesand disorders; neurological and neurodegenerative diseases anddisorders; fever associated with various diseases, disorders orconditions; incontinence; inflammatory diseases and disorders such asinflammatory bowel disease and Crohn's disease; respiratory diseases anddisorders such as chronic cough, asthma and chronic obstructivepulmonary disease (COPD); digestive disorders such as ulcers and acidreflux; metabolic diseases and disorders including obesity and diabetes;liver and kidney diseases and disorders; malignancies including cancers;aging-related disorders; and sensitivity to pain and touch.

Additional diseases or conditions that can be treated includeATP-related diseases or disorders including epilepsy, cognition, emesis,pain (e.g., migraine), asthma, peripheral vascular disease,hypertension, immune and inflammatory conditions, irritable bowelsyndrome, cystitis, depression, aging-associated degenerative diseases,urinary incontinence, premature ejaculation, cystic fibrosis, diabetes,contraception and sterility, and wound healing (see, for example,Foresta et al. (1992) J. Biol. Chem. 257:19443-19447; Wang et al. (1990)Biochim. Biophys. Res. Commun. 166:251-258; Burnstock and Williams,(2000) J. Pharmacol. Exp. Ther. 295: 862-869; and Burnstock, PharmacolRev (2006) 58:58-86).

TRPA1 inhibitors described herein can be used in the treatment of any ofthe foregoing or following diseases or conditions, including in thetreatment of pain associated with any of the foregoing or followingdiseases or conditions. When used in a method of treatment, an inhibitorcan be selected and formulated based on the intended route ofadministration. Inhibitors can be used to treat the underlying diseaseor condition, or to relieve a symptom of the disease or condition.Exemplary symptoms include pain associated with a disease or condition.

a. Sensitivity to Pain and Touch, or Pain-Related Diseases or Disorders

Compositions and methods provided herein may be used in connection withprevention or treatment of pain or sensitivity to pain and touch. Painor sensitivity to pain and touch may be indicated in a variety ofdiseases, disorders or conditions, including, but not limited to,diabetic neuropathy, breast pain, psoriasis, eczema, dermatitis, burn,post-herpetic neuralgia (shingles), nociceptive pain, peripheralneuropathic and central neuropathic pain, chronic pain, cancer and tumorpain, spinal cord injury, crush injury and trauma induced pain,migraine, cerebrovascular and vascular pain, sickle cell disease pain,rheumatoid arthritis pain, musculoskeletal pain including treating signsand symptoms of osteoarthritis and rheumatoid arthritis, orofacial andfacial pain, including dental, temperomandibular disorder, and cancerrelated, lower back or pelvic pain, surgical incision related pain,inflammatory and non-inflammatory pain, visceral pain, psychogenic painand soft tissue inflammatory pain, fibromyalgia-related pain, and reflexsympathetic dystrophy, and pain resulting from kidney stones or urinarytract infection. The compounds and methods of the invention may be usedin the treatment of chronic, as well as acute pain. Chronic or acutepain may be the result of injury, age, or disease.

Other ion channels have been implicated in reception or transmission ofpain. For example, the involvement of N-type calcium channels in thesynaptic transmissions that convey pain signals from sensory afferentnerve cells to the central nervous system has been recognized. Certainnaturally occurring peptide neurotoxins that specifically block N-typecalcium channel have been shown to act as extremely potent and efficientanalgesics in a wide range of animal pain models, including models ofinflammatory and neuropathic pain. The available evidence suggests thatN-type calcium channel blockers are at least as efficacious as opiates,are devoid of a number of the typical opiate side effects (e.g.respiratory depression) and that the analgesic effect is not subject totolerance development.

It has also been shown that potent peripheral analgesia induced by5-alpha-reduced neurosteroid is mediated in part by effects on T-typeCa²⁺ channels (Pathirathna et al., Pain. 2005 April; 114(3):429-43).

Ethosuximide, an anti-epileptic and relatively selective T-type calciumchannel blocker, has also been shown as being highly effective inreversing neuropathic pain caused by the commonly employed cytotoxicspaclitaxel or vincristine (Flatters and Bennett, Pain. 2004 May;109(1-2):150-61).

Pregabalin, a new drug that interacts with the alpha(2)-delta proteinsubunit of the voltage-gated calcium channel, is an efficacious and safetreatment for the pain of diabetic neuropathy (Richter et al., J Pain.2005 April; 6(4):253-60).

The foregoing demonstrate the involvement of various non-TRP channels inthe reception or transmission of pain. Specifically, the foregoingdemonstrate the involvement of various calcium channels in pain.

The present invention provides methods for treating pain that includeadministration of (i) antagonists of a TRPA1 function; (ii) combinationsof selective antagonists of a TRPA1 function and selective antagonistsof TRPV1 and/or TRPV3 function; or (iii) a pan-TRP inhibitor thatinhibits a function of two or more of TRPA1, TRPV1, and TRPV3.

In addition to TRPV family members, other TRP channels have beenimplicated in pain reception and/or sensation. For example, certain TRPMchannels including TRPM8 have been implicated in the reception and/orsensation of pain. Accordingly, in certain embodiments, the methods ofthe present invention include treating pain by administering (i) acombination of a selective TRPA1 antagonist and a selective TRPM8antagonist; (ii) a combination of a selective TRPA1 antagonist, aselective TRPM8 antagonist, and one or more of a selective TRPV1 and/orTRPV3 antagonist; (iii) a cross-TRP inhibitor that antagonizes afunction of TRPA1 and TRPM8; or (iv) a pan inhibitor that antagonizes afunction of TRPA1, TRPM8, and one or more of TRPV1 and TRPV3.

Without being bound by theory, we propose one possible mechanism for howa TRPA1 antagonist may help reduce pain. TRPA1 antagonists can lead tohyperpolarization of the cell. This may lead to a reduction in thefiring of neurons and/or a decrease in action potential frequency. Inaddition, TRPA1 inhibitors may reduce calcium influx into injured cellsand could prevent the calcium dependent changes in gene expression thatsometimes accompany injury.

b. Dermatological Diseases or Disorders

Influx of calcium across plasma membrane of skin cells is a criticalsignaling element involved in cellular differentiation in the skinepidermis (Dotto, 1999 Crit Rev Oral Biol Med 10:442-457). Regulating ormodulating the calcium entry pathway, and thus a critical control pointfor skin cell growth, can treat or prevent skin diseases or disordersthat are characterized by epidermal hyperplasia, a condition in whichskin cells both proliferate too rapidly and differentiate poorly. Suchdiseases include psoriasis, and basal and squamous cell carcinomas.Psoriasis, estimated to affect up to 7 million Americans, afflictssufferers with mild to extreme discomfort, enhanced susceptibility tosecondary infections, and psychological impact due to disfigurement ofthe affected areas (Lebwohl and Ali, 2001 J Am Acad Dermatol45:487-498). Basal cell carcinomas (BCC) and squamous cell carcinomas(SCC) of the skin represent at least one-third of all cancers diagnosedin the United States each year. More than 1 million new cases arereported annually and incidence is increasing. Despite being relativelynon-aggressive, slow-growing cancers, BCCs are capable of significantlocal tissue destruction and disfigurement. SCCs are more aggressive andthus present even greater complications. Further, given that 80% oflesions are on the head and neck with another 15% on shoulders, back orchest, BCCs and SCCs of the skin can have a significant impact on theappearance and quality of life of the afflicted patient.

Many dermatological disorders are accompanied by itch (pruritus).Pruritus and pain share many mechanistic similarities. Both areassociated with activation of C-fibers, both are potentiated byincreases in temperature and inflammatory mediators and both can bequelled with opiates. Decreasing neuronal excitability, particularlyC-fiber excitability may alleviate pruritus associated with dialysis,dermatitis, pregnancy, poison ivy, allergy, dry skin, chemotherapy andeczema.

c. Neurological or Neurodegenerative Diseases and Disorders

Neurodegenerative diseases and disorders include but are not limited toAlzheimer's disease (AD), Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis (ALS), and other brain disorders caused bytrauma or other insults including aging.

Mechanisms associated with calcium signaling may be altered in manyneurodegenerative diseases and in disorders resulting from brain injury.For example, fibroblasts or T-lymphocytes from patients with AD haveconsistently displayed an increase in Ca²⁺ release from intracellularstores compared to controls (Ito et al. (1994) Proc. Natl. Acad. Sci.U.S.A. 91:534-538; Gibson et al. (1996) Biochem. Biophys. ACTA1316:71-77; Etchenberrigaray et al. (1998) Neurobiology of Disease,5:37-45). Consistent with these observations, mutations in presenilingenes (PS1 or PS2) associated with familial AD (FAD) have been shown toincrease InsP3-mediated Ca²⁺ release from internal stores (Guo et al.(1996) Neuro Report, 8:379-383; Leissring et al. (1999) J.Neurochemistry, 72:1061-1068; Leissring et al. (1999) J. Biol. Chem.274(46):32535-32538; Leissring et al. (2000) J. Cell Biol.149(4):793-797; Leissring et al. (2000) Proc. Natl. Acad. Sci. U.S.A.97(15):8590-8593). Furthermore, mutations in PS1 or PS2 associated withan increase in amyloidogenic amyloid β peptide generation in AD arereported to be associated with a decrease in intracellular calcium level(Yoo et al. (2000) Neuron, 27(3):561-572).

Experimental traumatic brain injury has been shown to initiate massivedisturbances in Ca²⁺ concentrations in the brain that may contribute tofurther neuronal damage. Intracellular Ca²⁺ may be elevated by manydifferent ion channels. It has been further shown that channel blockersmay be beneficial in the treatment of neurological motor dysfunctionwhen administered in the acute posttraumatic period (Cheney et al.(2000) J. Neurotrauma, 17(1):83-91).

d. Inflammatory Diseases and Disorders

Compositions and methods provided herein may also be used in connectionwith treatment of inflammatory diseases. These diseases include but arenot limited to asthma, chronic obstructive pulmonary disease, rheumatoidarthritis, osteoarthritis, inflammatory bowel disease,glomerulonephritis, neuroinflammatory diseases such as multiplesclerosis, and disorders of the immune system.

The activation of neutrophils (PMN) by inflammatory mediators is partlyachieved by increasing cytosolic calcium concentration ([Ca²⁺]_(i)).Certain calcium channel-mediated calcium influx in particular is thoughtto play an important role in PMN activation. It has been shown thattrauma increases PMN store-operated calcium influx (Hauser et al. (2000)J. Trauma Injury Infection and Critical Care 48 (4):592-598) and thatprolonged elevations of [Ca²⁺]_(i) due to enhanced store-operatedcalcium influx may alter stimulus-response coupling to chemotaxins andcontribute to PMN dysfunction after injury. Modulation of PMN [Ca²⁺]_(i)through store-operated calcium channels might therefore be useful inregulating PMN-mediated inflammation and spare cardiovascular functionafter injury, shock or sepsis (Hauser et al. (2001) J. Leukocyte Biology69 (1):63-68).

Peripheral neuropathy, for example diabetic neuropathy, is a particularcondition that involves both a neuronal and an inflammatory component.Without being bound by a mechanistic theory, the TRPA1 antagonists ofthe invention may be useful in treating peripheral neuropathiesincluding, but not limited to, diabetic neuropathy. In addition to theiruse in the treatment of peripheral neuropathies (e.g., reducinginflammation), the subject inhibitors may also be useful in reducing thepain associated with peripheral neuropathy.

Neurogenic inflammation often occurs when neuronal hyperexcitabilityleads to the release of peptides that trigger inflammation. Thesepeptides include substance P and CGRP. Blocking TRPA1 would reduceneuronal activity and thus could block neurogenic inflammation.

e. Cancer and Other Proliferative Diseases

Compositions and methods provided herein may also be used in connectionwith treatment of malignancies, including, but not limited to,malignancies of lymphoreticular origin, bladder cancer, breast cancer,colon cancer, endometrial cancer, head and neck cancer, lung cancer,melanoma, ovarian cancer, prostate cancer and rectal cancer, in additionto skin cancers described above. Intracellular calcium level may play animportant role in cell proliferation in cancer cells (Weiss et al.(2001) International Journal of Cancer 92 (6):877-882).

In addition, pain associated with cancer or with cancer treatment is asignificant cause of chronic pain. Cancers of the bone, for example,osteosarcoma, are considered exceptionally painful, and patients withadvanced bone cancer may require sedation to tolerate the intense andpersistent pain. Accordingly, TRPA1 antagonists of the inventionrepresent a significant possible therapeutic for the treatment of pain,for example, the pain associated with cancer or with cancer treatment.

Given that TRPA1 is differentially expressed in transformed cells, TRPA1blockers may also affect the proliferation of transformed cells and thusbe a useful way to slow the disease (see Jaquemar et al. (1999) JBC274(11): 7325-33). Thus TRPA1 antagonists could alleviate both the causeand symptoms of cancer pain.

Cancer treatments are not only painful, but they may even be toxic tohealthy tissue. Some chemotherapeutic agents can cause painfulneuropathy. Accordingly, TRPA1 antagonists of the invention represent asignificant possible therapeutic for the treatment of the pain and/orinflammation associated with cancer treatments that cause neuropathy.

A major function of prostaglandins is to protect the gastric mucosa.Included in this function is the modulation of intracellular calciumlevel in human gastric cells which plays a critical role in cellproliferation. Consequently, inhibition of prostaglandins bynonsteroidal anti-inflammatory drugs (NSAIDs) can inhibit calcium influxin gastric cells (Kokoska et al. (1998) Surgery (St Louis) 124(2):429-437). The NSAIDs that relieve inflammation most effectively alsoproduce the greatest gastrointestinal damage (Canadian Family Physician,January 1998, p. 101). Thus, the ability to independently modulatecalcium channels in specific cell types may help to alleviate such sideeffect of anti-inflammatory therapy. Additionally or alternatively,administration of TRPA1 inhibitory compounds of the present inventionmay be used in combination with NSAIDs, thus promoting pain relief usingreduced dosage of NSAIDs.

f. Incontinence

Incontinence is a significant social and medical problem affecting bothmen and women. Incontinence has many causes including, but not limitedto, age, pregnancy, radiation exposure, surgery, injury, cancer,enlargement of the prostatic, prostatic hyperplasia, and diseases of thebladder or musculature that supports the urethra. The inventioncontemplates methods for treating incontinence due to any of theforegoing, as well as incontinence of unknown cause or continence due toanxiety, stress, or depression.

Compositions and methods provided herein may be useful in connectionwith the treatment of incontinence. Animal models of incontinence areoften associated with an increase in the frequency of spontaneous actionpotentials and a chronic depolarization of the smooth muscle cells.Evidence suggests that a non-selective cation current could lead to thisdepolarization. Since TRPA1 mRNA is expressed in neurons that innervatebladder, blocking TRPA1 might be an effective treatment forincontinence. In addition, TRPA1 is activated by stimulation of themuscarinic type 1 acetylcholine receptor (M1, see Jordt et al. (2004)Nature 427:260-265). Antimuscarininc agents are well known drugs for thetreatment of condition such as overactive bladder. Thus blocking TRPA1,a downstream target of the M1 receptor might alleviate such conditionswithout the side effects that are associated with muscarinicantagonists.

Incontinence can be caused by any of a number of injuries, diseases, andconditions. Some of these may cause significant discomfort and pain, inaddition to the inconvenience and embarrassment of the incontinenceitself. For example, bladder cystitis is a painful condition that canalso lead to incontinence. For injuries or conditions resulting in bothincontinence and pain, TRPA1 inhibitors can be used to treat theincontinence, as well as to relieve the accompanying pain.

For embodiments in which a TRPA1 inhibitor is used to treatincontinence, the invention contemplates additional possible routes ofadministration. For example, in certain embodiments, the TRPA1 inhibitorcan be administered directly to the urethra or bladder via a catheter orother intraluminal device. However, in other embodiments, the TRPA1inhibitor can be administered orally, intravenously, subcutaneously,etc.

g. Temperature Regulation

Because of the effects of ion flux on arterial tension and relaxation,the subject compounds can also be used to affect thermal sensitivity.Furthermore, given that TRPA1 channels are thermal responsive channelsinvolved in the reception and sensation of cold stimuli, TRPA1antagonists can be used to modulate the sensation of cool, cold anddecreased temperatures that often accompany pain.

h. Hypertension

Blockers of voltage-gated calcium channels belong to a class ofmedications originally developed to treat hypertension. Such blockersinhibit the movement of calcium into the muscle cells of the heart andarteries. Because calcium is needed for these muscles to contract, suchblockers lower blood pressure by decreasing the force of cardiaccontractile response and relaxing the muscle walls of the arteries.Although TRPA1 is not a voltage-gated calcium channel, it is stillinstrumental in regulating calcium homeostasis, as well as the balanceof other ions, in cells and tissues. Accordingly, TRPA1 antagonists ofthe invention may be used to treat hypertension. Additional uses of thesubject compounds include other conditions that may be ameliorated, inwhole or in part, by relaxing the muscle walls of blood vessels.Exemplary conditions include headaches and migraine attacks.

As outlined above, compounds that antagonize a function of TRPA1 can beused in the treatment of many diseases, injuries, disorders, andconditions. In certain embodiments, TRPA1 inhibitors can be used in thetreatment of pain. As outlined above, TRPA1 inhibitors can be used inthe treatment of pain resulting from injury or disease, as well as painexperienced as a consequence of treatment. Exemplary classes of paininclude nociceptive pain, inflammatory pain, and neuropathic pain. Suchpain can be chronic or acute. TRPA1 inhibitors can be used in thetreatment of one or more of any of the foregoing classes of pain. Incertain embodiments, TRPA1 inhibitors can be used in the treatment ofnociceptive pain. In certain other embodiments, TRPA1 inhibitors can beused in the treatment of inflammatory pain. In certain otherembodiments, TRPA1 inhibitors can be used in the treatment ofneuropathic pain.

As outlined above, TRPA1 inhibitors may be particularly useful in thetreatment of pain associated with cancer, osteoarthritis, rheumatoidarthritis, post-herpetic neuralgia, burns, and other indicationsdetailed above. To further illustrate, additional exemplary indicationsfor which compounds of the present invention can be used include oralpain, Fabry's disease, complex regional pain syndrome, pancreatitis, andfibromyalgia syndrome.

Fabry's Disease

Vague complaints of pain in hands and feet may be a presenting feature.These symptoms are called acroparesthesias, as they reflect theperipheral neuropathy that is a frequent manifestation of the disease.This pain may be both episodic and chronic. Acute episodes may betriggered by exposure to extremes of temperature, stress, emotion,and/or fatigue.

Fibromyalgia

Fibromyalgia (FMS; fibromyalgia syndrome) is a widespreadmusculoskeletal pain and fatigue disorder. Fibromyalgia is characterizedby pain in the muscles, ligaments, and tendons. The condition affectsmore women than men, and occurs in people of all ages. Overall, FMS isestimated to afflict 3-6% of the population.

Patients have described the pain associated with fibromyalgia as deepmuscular aching, throbbing, shooting, and stabbing. The pain sometimesincludes an intense burning sensation. The pain and stiffness are oftenworse in the morning or after repetitive use of a particular musclegroup.

Additionally, varying levels of fatigue ranging from mild toincapacitating are often associated with fibromyalgia. Other symptoms offibromyalgia include gastrointestinal symptoms. Irritable bowel syndromeand IBS-like symptoms such as constipation, diarrhea, frequent abdominalpain, abdominal gas, and nausea occur in roughly 40 to 70% of FMSpatients. Acid reflux or gastroesophogeal reflux disease (GERD) occursat a similar frequency.

Another frequent and debilitating symptom of FMS is chronic headaches,including migraine and tension-type headaches. Such headaches areexperienced by approximately 70% of FMS patients. Additionally, FMSpatients often experience temporomandibular joint dysfunction syndrome(also known as TMJ) which produces pain in the jaw, teeth, and mouth.TMJ may also exacerbate headaches.

Other common symptoms of FMS include, but are not limited to,premenstrual syndrome and painful periods; chest pain; morningstiffness; cognitive or memory impairment; numbness and tinglingsensations; muscle twitching; irritable bladder; the feeling of swollenextremities; skin sensitivities; dry eyes and mouth; dizziness; andimpaired coordination. Additionally, patients are often sensitive toodors, loud noises, and bright lights.

The cause of FMS remains unknown. However, the onset of the disorder hasbeen linked to infections (viral or bacterial), rheumatoid arthritis,lupus, and hypothyroidism. The link between these and other possibletriggers is unclear.

The impact of FMS on the patient is directly correlated with the levelof pain and fatigue. Pain may be so severe as to interfere with normalwork or family functioning. There is currently no cure for FMS, andcurrent therapies focus primarily on improving sleep (to decreasefatigue) and treating pain. Compounds of the present invention could beused to help manage the pain associated with FMS. Such pain includes,but is not limited to, oral pain in the jaw, teeth, and mouth. Such painalso includes non-oral musco-skeletal pain, pain due to headaches, andpain due to gastrointestinal symptoms.

Complex Regional Pain Syndrome (CRPS; also known as chronic regionalpain syndrome) is a chronic pain condition. CRPS was formerly known asreflex sympathetic dystrophy (RSD). CRPS is a chronic, painful, andprogressive neurological condition that affects skin, muscles, joints,and bones. The syndrome usually develops in an injured limb, such as abroken leg or following surgery. However, many cases involve only aminor injury, such as a sprain, and sometimes no precipitating injuriousevent can be identified. CRPS involves continuous, intense pain that isdisproportionate to the severity of the injury. The pain worsens, ratherthan improves, over time.

Although CRPS can affect a variety of regions of the body, it most oftenaffects the arms, legs, hands, or feet. Often the pain begins in oneportion of a limb, but spreads over time to include the entire limb oreven to include a different limb. Typical features include dramaticchanges in the color and temperature of the skin over the affected limbor body part, accompanied by intense burning pain, skin sensitivity,sweating, and swelling.

Generally, CRPS is characterized into two categories. Type I occurs inthe absence of a precipitating nerve injury—although there may have beensome other type of precipitating injury. Type II (formerly calledcausalgia) occurs following a nerve injury. These categories are merelydescriptive, and do not correlate with symptomology or prognosis.

The National Institute of Neurological Disorders and Strokes (NINDS)reports that 2% to 5% of peripheral nerve injury patients and 12% to 21%of patients with paralysis on one side of the body (hemiplegia) developreflex sympathetic dystrophy as a complication. The Reflex SympatheticDystrophy Syndrome Association of America (RSDSA) reports that thecondition occurs following 1-2% of bone fractures.

Precipitating events associated with the onset of CRPS include thefollowing: cerebral lesions, heart disease, heart attack, infection,paralysis on one side of the body (hemiplegia), radiation therapy,repetitive motion disorder (e.g., carpal tunnel syndrome), spinal corddisorders, surgery, and trauma (e.g., bone fracture, gunshot, caraccident). However, in 10-20% of cases, no precipitating event can befound. Note that the injury that precedes the onset of CRPS may or maynot be significant.

The symptoms of CRPS may progress in three stages. An acute stage occursduring the first 1-3 months and may include burning pain, swelling,increased sensitivity to touch, increased hair and nail growth in theaffected region, joint pain, and color and temperature changes. Adystrophic stage may involve constant pain and swelling. The effectedlimb often feels cool to the touch and looks bluish. There is typicallymuscle stiffness and wasting (atrophy), as well as early bone loss(osteoporosis). These symptoms usually occur 3-6 months afterdevelopment of the disorder. During an atrophic stage, the skin becomescool and shiny, increased muscle stiffness and weakness occur, andsymptoms may spread to another limb.

Other symptoms include: burning pain, extreme sensitivity to touch, skincolor changes (red or bluish), skin temperature changes (hot or cold),joint pain, swelling (edema), frequent infections, muscle stiffness,muscle spasm, tremor, weakness, dermatitis, eczema, excessive sweating,and migraine headache. A TRPA1 inhibitor can be useful not only intreating the pain associated with CRPS, but also in relieving many ofthese other symptoms including dermatitis, eczema, and migraines.

Patients with CRPS often suffer from depression and anxiety due to theimpact of the disease of their quality of life.

There is currently no cure for CRPS, and thus treatment typically aimsto relieve painful symptoms. Doctors may prescribe topical analgesics,antidepressants, corticosteroids, and opioids to relieve pain. However,to this point, no single drug or combination of drugs has producedconsistent long-lasting improvement in symptoms. Other treatments mayinclude physical therapy, sympathetic nerve block, spinal cordstimulation, and intrathecal drug pumps to deliver opioids and localanesthetic agents via the spinal cord.

The goals of treatment are to control pain and to maintain as muchmobilization of the affected limb as possible. An individualizedtreatment plan is designed, which often combines treatment modalities.Currently, physical therapy, medications, nerve blocks, and psychosocialsupport are used. TRPA1 inhibitors according to the present inventioncan be used instead of or in addition to one or more of the currenttreatment modalities. For example, a TRPA1 inhibitor can be used as analternative to current medications, but combined with physical therapy.

TRPA1 inhibitors provide an alternative for managing pain in CRPSpatients. TRPA1 inhibitors may be used in combination with any of thecurrent medications used to treat CRPS patients. Alternatively, TRPA1inhibitors may be used as an alternative medication.

In addition to drug therapy, CRPS patients often receive physicaltherapy. TRPA1 inhibitors can be used in addition to physical therapy.Physical therapy may be important for helping retain range of motion andfunction in the affected limb. Appropriate pain management, for exampleusing a TRPA1 inhibitor, not only increases patient comfort, but alsofacilitates involvement in physical therapy.

Regardless of the particular combination of therapies used to managepain in CRPS patients, psychological support is often critical. TRPA1inhibitors can be used in combination with psychological support.

TRPA1 inhibitors of the present invention may be used in the treatmentof CRPS. For example, TRPA1 inhibitors of the present invention may beused to help relieve the pain associated with CRPS. TRPA1 inhibitors canbe used alone or as part of an overall treatment regimen to help managethe pain and other symptoms associated with CRPS. Pain management forCRPS sufferers is critical for maintaining a meaningful quality of life.Furthermore, effective pain management may allow sufferers toparticipate in physical therapy to help retain mobility and use of theeffected limbs.

Pancreatitis is an inflammation of the pancreas. The pancreas is a largegland behind the stomach and close to the duodenum. Normally, digestiveenzymes do not become active until they reach the small intestine, wherethey begin digesting food. But if these enzymes become active inside thepancreas, they start “digesting” the pancreas itself.

Acute pancreatitis occurs suddenly, lasts for a short period of time,and usually resolves. Chronic pancreatitis does not resolve itself andresults in a slow destruction of the pancreas. Either form can causeserious complications including bleeding, tissue damage, and infection.

Acute pancreatitis can be a severe, life-threatening illness with manycomplications. About 80,000 cases occur in the United States each year,and approximately 20 percent of these cases are characterized as severe.

Acute pancreatitis is usually, although not exclusively, caused bygallstones or by alcohol abuse. Acute pancreatitis usually begins withpain in the upper abdomen that may last for a few days. The pain may besevere and may become constant. The pain may be isolated to the abdomenor it may reach to the back and other areas. Sometimes, and for somepatients, the pain is sudden and intense. Other times, or for otherpatients, the pain begins as a mild pain that worsens after eating.Someone with acute pancreatitis often looks and feels very sick. Othersymptoms may include swollen and tender abdomen, nausea, vomiting,fever, and rapid pulse. Severe cases of acute pancreatitis may causedehydration and low blood pressure, and may even lead to organ failure,internal bleeding, or death.

During acute pancreatitis attacks, the blood levels of amylase andlipase are often increased by at least 3-fold. Changes may also occur inblood levels of glucose, calcium, magnesium, sodium, potassium, andbicarbonate.

The current treatment depends on the severity of the attack. Treatment,in general, is designed to support vital bodily functions, manage pain,and prevent complications. Although acute pancreatitis typicallyresolved in a few days, pain management during an attack is oftenrequired. TPRV3 inhibitors can be used to relieve the pain associatedwith acute pancreatitis.

Chronic pancreatitis—If injury to the pancreas continues, chronicpancreatitis may develop. Chronic pancreatitis occurs when digestiveenzymes attack and destroy the pancreas and nearby tissues, causingscarring and pain. Chronic pancreatitis may be caused by alcoholism, orby blocked, damaged, or narrowed pancreatic ducts. Additionally,hereditary factors appear to influence the disease, and in certaincases, there is no identifiable cause (so called idiopathicpancreatitis).

Most people with chronic pancreatitis have abdominal pain. The pain mayget worse when eating or drinking, spread to the back, or becomeconstant and disabling. Other symptoms include nausea, vomiting, weightloss, and fatty stools.

Relieving pain is the first step in treating chronic pancreatitis. Oncethe pain has been managed, a high carbohydrate and low fat dietary planis put in place. Pancreatic enzymes may be used to help compensate fordecrease enzyme production from the injured pancreas. Sometimes insulinor other drugs are needed to control blood glucose.

Although pain is typically managed using drug therapy, surgery may benecessary to relieve pain. Surgery may be necessary to drain an enlargedpancreatic duct or even to removing a portion of a seriously injuredpancreas.

Pain is frequently present with chronic pancreatitis. For example, painis present for approximately 75% of patients with alcoholic chronicpancreatitis, 50% of patients with late-onset idiopathic chronicpancreatitis, and 100% of patients with early-onset idiopathic chronicpancreatitis (DiMagno, 1999, Gastroenterology 116(5): 1252-1257).

A minority of patients with pain have readily identifiable lesions whichare relatively easy to treat surgically or endoscopically. In otherpatients, pain is often thought to result from a variety of causes,including elevated intrapancreatic pressure, ischemia, and fibrosis.Without being bound by theory, however, these phenomena are not likelythe underlying cause of the pain. Rather, pain may result from abackground of neuronal sensitization induced by damage to theperineurium and subsequent exposure of the nerves to mediators andproducts of inflammation.

Given the importance of effective pain management in patients withchronic pancreatitis, additional therapies for treating painful symptomsare important and useful. TRPA1 inhibitors can be used to manage thepain associated with chronic pancreatitis. TRPA1 inhibitors can be usedalone or as part of an overall therapeutic treatment plan to managepatients with chronic pancreatits. For example, TRPA1 inhibitors can beadministered with pancreatic enzymes and/or insulin as part of atherapeutic regimen designed to manage patients with chronicpancreatitis.

Oral pain is a particular category of pain that may be treated using theTRPA1 inhibitors of the present invention. The term “oral pain” refersto any pain in the mouth, throat, lips, gums, teeth, tongue, or jaw. Theterm is used regardless of the cause of the pain and regardless ofwhether the oral pain is a primary or secondary symptom of a particulardisease, injury, or condition.

Oral pain has a large number of possible causes. In certain embodiments,oral pain is caused by an injury or disease of the mouth, jaw, teeth,gums, throat, lips, or tongue. In certain other embodiments, oral painis a consequence of an injury or disease that primarily affects anotherpart of the body. In still other embodiments, oral pain is a side effectof a therapy used to treat an injury or disease of the mouth or anotherpart of the body. TRPA1 inhibitors are useful in treating oral painregardless of its cause.

All pain has a serious negative impact on the health and well being ofthe sufferer. However, oral pain may have a particularly deleteriousimpact on patient health and quality of life. In particular, oral paincan interfere with appropriate eating and drinking. Thus, individualswith oral pain are susceptible to weight loss, malnutrition, anddehydration. In some instances, oral pain may interfere with hydrationand nutrition so significantly as to require intravenous, nasogastric,or other artificial support (e.g., tube feeding and/or hydration).Additionally, oral pain can interfere with proper oral hygiene. Poororal hygiene may further exacerbate many of the causes of oral pain, forexample, oral pain due to infection or abscess.

In certain embodiments, oral pain is caused by ulcers, sores, or otherlesions in the mouth. For example, oral pain may be caused by ulcers,sores, or other lesions on the tongue, gums, lips, throat, or othertissues of the mouth. Alternatively or additionally, oral pain may becaused by inflammation of the throat, tongue, gums, lips, or othertissues of the mouth. Inflammation may accompany ulcers or otherlesions, or inflammation may occur prior to or in the absence offormation of ulcers or other lesions.

The invention contemplates treatment of oral pain by administering aTRPA1 inhibitor by any route of administration described herein. Incertain embodiments, TRPA1 inhibitors for use in the treatment of oralpain are administered orally. Preferred preparations for oraladministration of TRPA1 inhibitors for use in treating oral pain are asa mouthwash, a gel, a tooth paste or other paste, a liquid, a lozenge,via a swab, or in association with a mouth guard or dental apparatus.The preparation and particular method of administration will depend onthe cause of the oral pain, the overall health and underlying medicalconditions of the patient, the severity of the pain, and othermedications or therapies the patient is concurrently receiving. Amedical practitioner can readily determine the optimal formulation foruse in a particular patient.

The conditions provided below are intended to illustrate the range ofinjuries and diseases of diverse etiology that may lead to oral pain.The invention contemplates administration of a TRPA1 inhibitor,according to the present invention, to treat or prevent oral pain. Incertain embodiments, compounds of the invention can be orallyadministered, for example as a gel, paste, mouth wash, or other oralpreparation, to help treat or prevent oral pain associated with anyinjury, disease, or condition. Regardless of the particular formulation,the invention contemplates administration by, for example, directapplication to the affected area of the mouth, rinsing of the entiremouth, via a swab, via a syringe, or on a mouth guard or other dentalapparatus.

For any of these conditions, the invention contemplates administrationof a TRPA1 inhibitor alone, or in combination with one or more othercompounds or treatment regimens appropriate for the particular injury orcondition.

Oral Mucositis

Oral mucositis, also known as stomatitis, is a common complication ofmany cancer treatments. Patients receiving systemic chemotherapy and/orlocal radiotherapy often develop extremely painful ulcers of the oralmucosa. This side effect is not limited to patients suffering fromcancers of the head and neck, but rather is a debilitating side effectafflicting approximately 40% of all chemotherapy patients (Preventionand Treatment of Oral Mucositis in Cancer Patients, 1998, Best Practice:2, pages 1-6.)

Oral mucositis is extremely painful. Additionally, oral mucositisinterferes with proper nutrition and hydration of cancer patients. Giventhe already compromised status of patients undergoing chemotherapyand/or radiotherapy, further interference with nutrition and hydrationmay seriously undermine patient health. Furthermore, these ulcerspresent an increased risk of infection. This risk is particularly acutein patients with compromised immune systems. Examples of patients atparticular risk of developing an opportunistic infection are patientswhose treatment included removal of one or more lymph nodes, patientswho previously received high-dose chemotherapy in preparation for a bonemarrow or stem cell transplant, and patients with an underlyingimmunosuppressive disorder (e.g., HIV or hepatitis).

Canker Sores

Canker sores, also known as aphthous ulcers (aphthae), may be relativelysmall and out-of-sight. However, they are often painful, persistent andannoying. Canker sores are shallow ulcers in the mouth that can makeeating and talking uncomfortable. They may occur on the tongue, softpalate, inside the cheek or lip, or at the base of the gums. Cankersores differ from cold sores in that they occur on the internal softtissues of the mouth and aren't contagious. Conversely, cold soresalmost always start out on the lips and don't often spread to the softtissues of the mouth. In addition, cold sores are caused by a form ofthe herpes virus, making them extremely contagious.

Researchers generally believe that stress or tissue injury may cause theeruption of canker sores. In some cases a minor injury, for examplebiting the inside of the mouth or eating rough foods, may trigger acanker sore. Other causes may include: (i) faulty immune systemfunction; (ii) nutritional problems, such as a deficiency of vitaminB-12, zinc, folic acid or iron; (iii) diseases of the gastrointestinaltract; (iv) food allergies; or (v) the menstrual cycle.

Canker sores can occur at any age, but often they first appear betweenthe ages of 10 and 40 years. Although canker sores typically resolve ontheir own, they can be very uncomfortable.

Dental/Tooth Abscess

Infection or decay can lead to an abscess. An abscess may have seriousdental and medical consequences. For example, a severe infection causedby a dental abscess may lead to a sinus or systemic infection.Furthermore, an abscess may lead to the need to extract one or moreteeth. Extraction may be necessary due to significant tooth decay, orbecause the infection is too severe to fully treat in the presence ofthe offending tooth.

Regardless of the ultimate outcome, a dental abscess may be extremelypainful. Not only is the pain uncomfortable, but it may interfere withproper nutrition and hydration. Methods and compositions for reducingthe pain associated with dental abscess would provide significantbenefits for their management.

Gastroesophageal Reflux Disease

Gastroesophageal reflux disease, or GERD, occurs when the loweresophageal sphincter (LES) does not close properly and stomach contentsleak back into the esophagus. The LES is a ring of muscle at the bottomof the esophagus that acts like a valve between the esophagus andstomach. When refluxed stomach acid touches the lining of the esophagus,it causes a burning sensation in the chest or throat. This is oftenexperienced as heartburn. The refluxed fluid may even be tasted in theback of the mouth, a sensation commonly referred to as acid indigestion.

Although occasional heartburn is uncommon and not necessarily indicativeof GERD, heartburn that occurs more than twice a week may be a sign ofGERD. In addition to the discomfort of heartburn and indigestion, GERDmay lead to other serious health problems. For example, over time, acidrefluxed to the back of the throat can lead to oral sores, lesions, orulcers in the mouth, gums, tongue, throat, or lips. The lesions cancause significant pain, can interfere with nutrition and hydration, andcan leave a person vulnerable to infection.

Administration of TRPA1 inhibitors, according to the present invention,may be useful in treating oral pain from lesions caused by GERD. TRPA1inhibitors may be used as part of a treatment regimen where the TRPA1inhibitor is administered to help manage the discomfort of the orallesion, while other agents or therapeutics interventions are used tomanage the GERD.

Gingivostomatitis

Gingivostomatitis is a disorder involving sores on the mouth and gumsthat result from a viral infection. Gingivostomatitis is characterizedby inflammation of the gums and mucosa and multiple oral ulcers. Theinflammation and ulcers are caused by viral infections, particularlythose that cause common childhood illness such as herpes virus (coldsores and acute herpetic stomatitis), and Coxsackie viruses (hand, footand mouth disease and herpangina). These viruses cause shallow ulcerswith a grayish or yellowish base and a slightly red margin, on thetissues of the gums (gingiva), the lining of the cheeks (buccal mucosa),or other soft tissues of the mouth. Although this condition can occur inpatients of any age, it is particularly common in children.

The oral ulcers caused by these viruses can be very painful. The ulcersare often accompanied by a fever. Overall, the condition can takeseveral weeks to resolve. The recognized treatments forgingivostomatitis focus on reducing the pain caused by the oral ulcers.This is particularly important for children who may refuse food orliquids because of their discomfort, thus making them especiallysusceptible to dehydration. Compounds of the present invention can beused to treat the pain associated with these oral ulcers.

Oral Thrush

Oral thrush is a fungal infection generally caused by the yeast fungus,Candida albicans, in the mucous membranes of the mouth. Strictlyspeaking, thrush is only a temporary Candida infection in the oralcavity of babies. However, the term is used generally to refer to fungalinfections in the mouths and throats of children and adults.

Candida is present in the oral cavity of almost half of the population.For example, everyone who wears dentures has Candida, withoutnecessarily suffering any ill effects. Generally, Candida does notcreate problems until there is a change in the chemistry of the oralcavity such that the growth of Candida is favored over the othermicroorganisms that typically inhabit the mouth and throat. Changes inoral chemistry sufficient to permit the growth of Candida may occur as aside effect to taking antibiotics or chemotherapeutics. Overall patienthealth may also influence the chemistry of the mouth. HIV infection,diabetes, malnutrition, age, and immunodeficiency are exemplaryconditions that can shift oral chemistry enough to permit the overgrowthof Candida in the mouth and throat.

In addition to shifts in oral chemistry, people whose dentures don't fitwell can sustain breaks in the mucous membranes in their mouth. Thesebreaks provide an opportunity for Candida infection in the mouth andlips.

Thrush causes white, cream-colored, or yellow spots in the mouth. Thespots are slightly raised. If these spots are scraped they tend tobleed. Thrush can be very uncomfortable, and may cause a burningsensation in the mouth and throat. The discomfort may interfere withhydration and nutrition. Furthermore, the discomfort may interfere withproper oral hygiene such as brushing and flossing.

Standard treatment of thrush is by administration of anti-fungal agents.These agents can be administered directly to the mouth, for example, inthe form of pastilles that are sucked or oral suspensions that are heldin the mouth before swallowing. Examples include nystatin (e.g., Nystanoral suspension), amphotericin (e.g., Fungilin lozenges) or miconazole(e.g., Daktarin oral gel). In addition to standard anti-fungal therapy,compounds of the present invention can be administered to manage thepain and discomfort associated with thrush.

Glossitis

Glossitis is an abnormality of the tongue that results frominflammation. Glossitis occurs when there is acute or chronicinflammation of the tongue. It causes the tongue to swell and changecolor. Finger-like projections on the surface of the tongue (papillae)are lost, causing the tongue to appear smooth. Glossitis has a number ofcauses including, but not limited to, the following: bacterialinfections; viral infections (including oral herpes simplex); injury ortrauma; exposure to irritants (e.g., tobacco, alcohol, hot foods,spices); allergic reactions; vitamin or mineral deficiencies (e.g., irondeficiency anemia, pernicious anemia and other B-vitamin deficiencies);or as a side effect of other diseases or disorders.

The symptoms of glossitis include swelling, soreness, and tenderness ofthe tongue. Additionally, the tongue often changes appearance, becomingsmooth and dark red in color. As a consequence of the swelling anddiscomfort, glossitis often makes chewing, swallowing, and speakingdifficult.

The typical treatment for glossitis depends on the underlying cause ofthe inflammation. Regardless of the particular antibiotics,anti-inflammatories, or anti-viral agents that may be administered tocombat the underlying cause of glossitis, compounds according to thepresent invention may be administered to decrease the pain anddiscomfort associated with glossitis. Decreasing the pain associatedwith glossitis is especially important when it interferes with propernutrition and hydration, or when it interferes with or prevents properoral hygiene.

Cutaneous Diseases

Oral ulcers may result from any of a number of cutaneous diseases. Forexample, lichen planus, pemphigus, pemphigoid, and erythema multiformemay lead to oral ulcers. Such oral ulcers may cause significant painthat can be treated using the compounds of the present invention.

Reduction of pain may help facilitate healing. This is especiallyimportant for patients with pemphigus and pemphigoid who develop oralulcers. Such patients are already immunosuppressed, and may thus be moresusceptible to opportunistic infections from lesions in the mouth.

Gastrointestinal Diseases

Oral ulcers may result from any of a number of gastrointestinaldiseases. Conditions which interfere with proper digestion, managementand flow of stomach and other digestive acids, motility, and eliminationmay lead to oral ulcers and other lesions. In some instances, the oralulcers are the results of acids or partially digested food refluxinginto the esophagus. In other instances, the oral ulcers result fromfrequent vomiting. In still other instances, oral ulcers occur due tovitamin deficiency, mineral deficiency or other nutritional deficiencysecondary to the gastrointestinal disease. In still other instances,oral ulcers are part of the complex etiology that characterizes thegastrointestinal disease.

Oral ulcers resulting from or experienced as part of a gastrointestinaldisease may be extremely painful. They may undermine proper nutritionand hydration for a patient whose underlying gastrointestinal diseasemay already impose multiple limitations on diet. Accordingly, methodsand compositions for decreasing the discomfort and pain associated withthese oral ulcers offer substantial benefits for patients with anunderlying gastrointestinal condition.

Exemplary gastrointestinal conditions which may lead to oralinflammation, lesions, or ulcers include, but are not limited to,Crohn's disease, ulcerative colitis, irritable bowel syndrome, celiacsprue, and dermatitis herpetiformis. The primary symptoms of theseconditions may be managed with diet, stress management, and medications.The TRPA1 inhibitors of the present invention may be used to help managethe pain and discomfort of oral inflammation, lesions, or ulcers causedby any of these gastrointestinal conditions.

Rheumatoid Diseases

A consequence of several rheumatoid diseases is oral ulcers. Forexample, lupus, Behcet's syndrome, Sweet's syndrome, and Reiter'sdisease may all lead to oral ulcers. Such oral ulcers may causesignificant mouth pain that can be treated using the compounds of thepresent invention.

Sjogren's Syndrome

Dry mouth is a common symptom associated with Sjögren's syndrome. Drymouth is caused by a decrease in the production of saliva. Saliva is anessential body fluid for protection and preservation of the oral cavityand oral functions. Although saliva is mostly water, it also containsover 60 substances which serve the following important functions:protect, lubricate and cleanse the oral mucosa; aid chewing, swallowingand talking; protect the teeth against decay; protect the mouth, teeth,and throat from infection by bacteria, yeasts, and viruses; support andfacilitate our sense of taste.

Given the important functions of saliva, decreased salivation can leadto many problems. If the condition persists for months or years, apatient may develop oral complications such as difficulty swallowing,severe and progressive tooth decay, oral infections (particularlyfungal), or combinations of these. Many of the conditions can causediscomfort, in their own right, and may also lead to oral lesions orulcers.

Several medications are available to help increase salivary secretion inpatients with dry mouth. Pilocarpine (Salagen®) and cevimeline (Evoxac®)reduce symptoms of dry mouth and increase salivary secretion. However,these drugs don't prevent tooth decay or treat the oral pain associatedwith the symptoms or effects of dry mouth. Compounds of the presentinvention can be used to treat the pain associated with dry mouth.

Vitamin or Mineral Deficiencies

In some instances, vitamin or mineral deficiencies may lead to ulcers orother sores in the mouth. For example, deficiency in vitamin C may leadto the oral lesions characteristic of scurvy. Deficiencies in vitaminsB1, B2, B6, or B12 may also lead to oral lesions. Additionally,deficiencies in zinc, folic acid, iron, selenium, or calcium may lead tooral lesions.

In certain embodiments, a vitamin or mineral deficiency is aprecipitating factor leading to a canker sore. However, a vitamin ormineral deficiency may also lead to other types of oral ulcers andlesions. Regardless of the nature of the lesion, compounds of thepresent invention can be used to help manage the associated pain.

Allergies

Allergies can sometimes lead to canker sores and other oral lesions.Oral lesions due to an allergy may be more likely when a person's oraltissues come into contact with the causative allergen. However, contactbetween the allergen and oral tissue is not necessarily required toproduce an oral lesion. Exemplary allergens that can lead to orallesions include food allergens such as fruits and vegetables (e.g.,strawberries, lemons, oranges, pineapples, apples, figs, tomatoes);shellfish; chocolate; nuts; dairy (e.g., milk and cheese); cereal grains(e.g., buckwheat, wheat, oats, rye, barley, gluten protein found ingrains); additives (e.g., cinnamonaldehyde (a flavoring agent), benzoicacid (a preservative); toothpastes (e.g., some people have a sensitivityto sodium laurel sulfate found in certain toothpastes and mouthwashes);nonsteroidal anti-inflammatory drugs (NSAIDs; some people have asensitivity leading to canker sores in response to this class of drug).

Other Exemplary Conditions and Injuries

The foregoing are merely exemplary of diseases and conditions that causeor lead to inflammation, lesions, ulcers, or other sources of oral pain.In other embodiments, the oral pain is due to an injury to the mouth,jaw, lips, gums, or teeth. In other embodiments, the oral pain is due tooral surgery, for example, surgery for cancer, tooth extraction, or jawremodeling. Other conditions that may lead to oral ulcers, and thus oralpain, include, but are not limited to chickpox, herpes zoster,infectious mononucleosis, syphilis, tuberculosis, acute necrotizinggingivitis, and burning mouth syndrome. Additionally, conditions thatlead to a compromised immune system put patients at risk for, amongother complications, oral inflammation, lesions, or ulcers. HIVinfection, AIDS, and hepatitis are all conditions that undermine theimmune system and may lead to oral lesions or ulcers. Additionally,individuals taking immunosuppressants (e.g., organ transplantrecipients, bone marrow recipients, stem cells recipients, patients withan autoimmune disease) are at increased risk of developing painful orallesions.

The invention contemplates the use of TRPA1 inhibitors, according to thepresent invention, in the treatment of oral pain regardless of theunderlying cause. In certain embodiments, TRPA1 inhibitors for treatingoral pain can be administered orally, for example, as a paste, mouthwash, gel, or other liquid preparation. In certain embodiments, thepaste, mouth wash, gel, or other liquid preparation is administered viaa swab, mouth guard, or other dental apparatus. In certain embodiments,the preparation is applied locally to the mouth, but is not otherwiseingested. For example, a mouth wash formulation that is not swallowedmay be used. Regardless of the formulation and route of administration,the invention contemplates administration of the subject TRPA1inhibitors as part of an overall treatment strategy that also includestherapies appropriate for the particular disease or condition thatcaused the oral inflammation, lesion, or ulcer.

TRPA1 inhibitors may be used to treat oral pain resulting from any ofthe foregoing injuries, diseases, or conditions. Additionally,Applicants note that the subject TRPA1 inhibitors may also be useful inthe treatment of the underlying aforementioned diseases and conditionsthemselves. Specifically, TRPA1 inhibitors may be useful in thetreatment of inflammation, and thus diseases or conditions with aninflammatory component, whether the symptoms manifest themselves in themouth or in other parts of the body, may themselves be treatable withthe subject TRPA1 inhibitors. Accordingly, the invention contemplatesand recognizes that for some conditions the therapeutic affects ofadministering a TRPA1 inhibitor may be two-fold: (i) decreasing painassociated with one or more symptoms of a disease or condition and (ii)treating the underlying symptoms or disease.

Disease and Injury Models

Compounds that antagonize TRPA1 function may be useful in theprophylaxis and treatment of any of the foregoing injuries, diseases,disorders, or conditions. In addition to in vitro assays of the activityof these compounds, their efficacy can be readily tested in one or moreanimal models. By way of example, numerous well known animal modelsexist. One or more suitable animal models (e.g., suitable in light ofthe particular indication) can be selected.

Pain can be generally categorized as chronic pain and acute pain. Thetwo categories of pain differ in duration, as well as underlyingmechanism. Chronic pain is not only persistent, but also does notgenerally respond well to treatment with currently available analgesics,non-steroidal anti-inflammatory drugs, and opioids.

Two broad sub-categories of chronic pain are neuropathic pain and cancerpain. Wang and Wang (2003) Advanced Drug Delivery Reviews 55: 949-965.Neuropathic pain refers to pain resulting from damage (e.g., fromdisease, injury, age) to the nervous system (e.g., nerves, spinal cord,CNS, PNS). Cancer-related pain may be caused by tumor infiltration,nerve compression, substances secreted by tumors, or the particulartreatment regimen (e.g., radiation, chemotherapeutics, surgery).

Pain is also often classified mechanistically as nociceptive,inflammatory, or neuropathic. Nociceptive pain is pain experiencedfollowing, for example, changes or extremes in temperature, exposure toacids, exposure to chemical agents, exposure to force, and exposure topressure. Reception of painful stimuli sends impulses to the dorsal rootganglia. The response is typically a combination of a reflexive response(e.g., withdrawal from the stimuli) and an emotional reaction.Inflammation is the immune system's response to injury or disease. Inresponse to injury or disease, macrophages, mast cells, neutrophils, andother cells of the immune system are recruited. This infiltration ofcells, along with the release of cytokines and other factors (e.g.,histamine, serotonin, bradykinin, prostaglandins, ATP, H+, nerve growthfactor, TNFα, endothelins, interleukins), can cause fever, swelling, andpain. Current treatments for the pain of inflammation include Cox2inhibitors and opioids. Neuropathic pain refers to pain resulting fromdamage (e.g., from disease, injury, age) to the nervous system (e.g.,nerves, spinal cord, CNS, PNS). Current treatment for neuropathic painincludes tricyclic antidepressants, anticonvulsants, Na+ channelblockers, NMDA receptor antagonists, and opioids.

There are numerous animal models for studying pain. The various modelsuse various agents or procedures to simulate pain resulting frominjuries, diseases, or other conditions. Blackburn-Munro (2004) Trendsin Pharmacological Sciences 25: 299-305 (see, for example, Tables 1, 3,or 4). Behavioral characteristics of challenged animals can then beobserved. Compounds or procedures that may reduce pain in the animalscan be readily tested by observing behavioral characteristics ofchallenged animals in the presence versus the absence of the testcompound(s) or procedure.

Exemplary behavioral tests used to study chronic pain include tests ofspontaneous pain, allodynia, and hyperalgesia. Id. To assess spontaneouspain, posture, gait, nocifensive signs (e.g., paw licking, excessivegrooming, excessive exploratory behavior, guarding of the injured bodypart, and self-mutilation) can be observed. To measure evoked pain,behavioral responses can be examined following exposure to heat (e.g.,thermal injury model).

Exemplary animal models of pain include, but are not limited to, theChung model, the carageenan induced hyperalgesia model, the Freund'scomplete adjuvant induced hyperalgesia model, the thermal injury model,the formalin model and the Bennett Model. The Chung model of neuropathicpain (without inflammation) involves ligating one or more spinal nerves.Chung et al. (2004) Methods Mol Med 99: 35-45; Kim and Chung (1992) Pain50: 355-363. Ligation of the spinal nerves results in a variety ofbehavioral changes in the animals including heat hyperalgesia, coldallodynia, and ongoing pain. Compounds that antagonize TRPA1 can beadministered to ligated animals to assess whether they diminish theseligation-induced behavioral changes in comparison to that observed inthe absence of compound.

Carageenan induced hyperalgesia and Freund's complete adjuvant (FCA)induced hyperalgesia are models of inflammatory pain. Walker et al.(2003) Journal of Pharmacol Exp Ther 304: 56-62; McGaraughty et al.(2003) Br J Pharmacol 140: 1381-1388; Honore et al. (2005) J PharmacolExp Ther. Compounds that antagonize TRPA1 can be administered tocarrageenan or FCA challenged animals to assess whether they diminishthermal hyperalgesia in comparison to that observed in the absence ofcompound. In addition, the ability of compounds that antagonize TRPA1function to diminish cold and/or mechanical hypersensitivity can also beassessed in these models. Typically, the carrageenan inducedhyperalgesia model is believed to mimic acute inflammatory pain and theCFA model is believed to mimic chronic pain and chronic inflammatorypain.

The Bennett model uses prolonged ischemia of the paw to mirror chronicpain. Xanthos et al. (2004) J Pain 5: S1. This provides an animal modelfor chronic pain including post-operative pain, complex regional painsyndrome, and reflex sympathetic dystrophy. Prolonged ischemia inducesbehavioral changes in the animals including hyperalgesia to mechanicalstimuli, sensitivity to cold, pain behaviors (e.g., paw shaking,licking, and/or favoring), and hyperpathia. Compounds that antagonizeTRPA1 can be administered to challenged animals to assess whether theydiminish any or all of these behaviors in comparison to that observed inthe absence of compound. Similar experiments can be conducted in athermal injury or UV-burn model which can be used to mimicpost-operative pain.

Migraines are associated with significant pain and inability to completenormal tasks. Several models of migraine exist including the ratneurogenic inflammation model, (see Buzzi et al (1990) Br J Pharmacol;99:202-206), and the Burstein Model (see Strassman et al., (1996) Nature384: 560-564).

Additional models of neuropathic pain include central pain models basedon spinal cord injury. Chronic pain is generated by inducing a spinalcord injury, for example, by dropping a weight on a surgically exposedarea of spinal cord (e.g., weight-drop model). Spinal cord injury canadditionally be induced by crushing or compressing the spinal cord, bydelivering neurotoxin, using photochemicals, or by hemisecting thespinal cord. Wang and Wang (2003).

Additional models of neuropathic pain include peripheral nerve injurymodels. The term peripheral neuropathy encompasses a variety ofdiseases, conditions, and injuries. One of skill in the art can readilyselect an appropriate model in light of the particular condition ordisease under investigation. Exemplary models include, but are notlimited to, the neuroma model, the Bennett model, the Seltzer model, theChung model (ligation at either L5 or L5/L6), the sciatic cryoneurolysismodel, the inferior caudal trunk resection model, and the sciaticinflammatory neuritis model. Id.

Exemplary models of inflammatory pain include the rat model ofintraplantar bradykinin injection. Briefly, the baseline thermalsensitivity of the animals is assessed on a Hargreave's apparatus. TRPA1blockers are then administered systemically. Bradykinin is subsequentlyinjected into the paw and a hyperalgesia is allowed to develop. Thermalescape latency is then measured at multiple time points over the nextfew hours (Chuang et al., 2001; Vale et al., 2004).

Exemplary models of neuropathic pain associated with particular diseasesare also available. Diabetes and shingles are two diseases oftenaccompanied by neuropathic pain. Even following an acute shinglesepisodes, some patients continue to suffer from postherpetic neuralgiaand experience persistent pain lasting years. Neuropathic pain caused byshingles and/or postherpetic neuralgia can be studied in thepostherpetic neuralgia model (PHN). Diabetic neuropathy can be studiedin diabetic mouse models, as well as chemically induced models ofdiabetic neuropathy. Wang and Wang (2003).

As outlined above, cancer pain may have any of a number of causes, andnumerous animal models exist to examine cancer pain related to, forexample, chemotherapeutics or tumor infiltration. Exemplary models oftoxin-related cancer pain include the vincristine-induced peripheralneuropathy model, the taxol-induced peripheral neuropathy model, and thecisplatin-induced peripheral neuropathy model. Wang and Wang (2003). Anexemplary model of cancer pain caused by tumor infiltration is thecancer invasion pain model (CIP). Id.

Primary and metastatic bone cancers are associated with tremendous pain.Several models of bone cancer pain exist including the mouse femur bonecancer pain model (FBC), the mouse calcaneus bone cancer pain model(CBC), and the rat tibia bone cancer model (TBC). Id.

An additional model of pain is the formalin model. Like the carrageenanand CFA models, the formalin model involves injection of an irritantintradermally or intraperitoneally into an animal. Injection offormalin, a 37 percent solution of formaldehyde, is the most commonlyused agent for intradermal paw injection (the formalin test). Injectionof a 0.5 to 15 percent solution of formalin (usually about 3.5%) intothe dorsal or plantar surface of the fore- or hindpaw produces abiphasic painful response of increasing and decreasing intensity forabout 60 minutes after the injection. Typical responses include the pawbeing lifted, licked, nibbled, or shaken. These responses are considerednociceptive. The initial phase of the response (also known as the EarlyPhase), which lasts 3 to 5 minutes, is probably due to direct chemicalstimulation of nociceptors. This is followed by 10 to 15 minutes duringwhich animals display little behavior suggestive of nociception. Thesecond phase of this response (also known as the Late Phase) startsabout 15 to 20 minutes after the formalin injection and lasts 20 to 40minutes, initially rising with both number and frequency of nociceptivebehaviors, reaching a peak, then falling off. The intensities of thesenociceptive behaviors are dependent on the concentration of formalinused. The second phase involves a period of sensitization during whichinflammatory phenomena occur. The two phases of responsiveness toformalin injection makes the formalin model an appropriate model forstudying mociceptive and acute inflammatory pain. It may also model, insome respects, neuropathic pain.

In addition to any of the foregoing models of chronic pain, compoundsthat antagonize TRPA1 function can be tested in one or more models ofacute pain. Valenzano et al. (2005) Neuropharmacology 48: 658-672.Regardless of whether compounds are tested in models of chronic pain,acute pain, or both, these studies are typically (though notexclusively) conducted, for example, in mice, rats, or guinea pigs.Additionally, compounds can be tested in various cell lines that providein vitro assays of pain. Wang and Wang (2003).

Many individuals seeking treatment for pain suffer from visceral pain.Animal models of visceral pain include the rat model of inflammatoryuterine pain (Wesselmann et al., (1997) Pain 73:309-317), injection ofmustard oil into the gastrointestinal tract to mimic irritable bowelsyndrome (Kimball et al., (2005) Am J Physiol Gastrointest LiverPhysiol, 288(6):G1266-73), injection of mustard oil into the bladder tomimic overactive bladder or bladder cystitis (Riazimand (2004), BJU 94:158-163). The effectiveness of a TRPA1 compound can be assessed by adecrease in writhing, gastrointestinal inflammation or bladderexcitability.

The foregoing animal models are relied upon extensively in the study ofpain. The following provide additional exemplary references describingthe use of these models in the study of pain: thermal injury model(Jones and Sorkin, 1998, Brain Res 810: 93-99; Nozaki-Taguchi and Yaksh,1998, Neuroscience Lett 254: 25-28; Jun and Yaksh, 1998, Anesth Analg86: 348-354), formalin model (Yaksh et al., 2001, J Appl Physiol 90:2386-2402), carrageenan model (Hargreaves et al., 1988, Pain 32: 77-88),and CFA model (Nagakura et al., 2003, J Pharmacol Exp Ther 306:490-497).

Inflammation is often an important contributing factor to pain. As such,it is useful to identify compounds that act as anti-inflammatories. Manycompounds that reduce neural activity also prevent neurogenicinflammation. To measure inflammation directly, the volume of a rat pawcan be assessed using a plethysmometer. After baseline measurement istaken, carrageenan can be injected into the paw and the volume can bemonitored over the course of hours in animals that have been treatedwith vehicle or drug. Drugs that reduce the paw swelling are consideredto be anti-inflammatory.

For testing the efficacy of TRPA1 antagonists for the treatment ofcough, experiments using the conscious guinea pig model of cough can bereadily conducted. Tanaka and Maruyama (2003) Journal Pharmacol Sci 93:465-470; McLeod et al. (2001) Br J Pharmacol 132: 1175-1178. Briefly,guinea pigs serve as a useful animal model for cough because, unlikeother rodents such as mice and rats, guinea pigs actually cough.Furthermore, guinea pig coughing appears to mimic human coughing interms of the posture, behavior, and appearance of the coughing animal.

To induce cough, conscious guinea pigs are exposed to an inducing agentsuch as citric acid or capsaicin. The response of the animal is measuredby counting the number of coughs. The effectiveness of a coughsuppressing agent, for example a compound that inhibits TRPA1, can bemeasured by administering the agent and assessing the ability of theagent to decrease the number of coughs elicited by exposure to citricacid, capsaicin, or other similar cough-inducing agent. In this way,TRPA1 inhibitors for use in the treatment of cough can be readilyevaluated and identified.

Additional models of cough include the unconscious guinea pig model.Rouget et al. (2004) Br J Pharmacol 141: 1077-1083. Either of theforegoing models can be adapted for use with other animals capable ofcoughing. Exemplary additional animals capable of coughing include catsand dogs.

Numerous rodent models of incontinence exist. These include models ofincontinence induced by nerve damage, urethral impingement andinflammation. Models of urethral impingement include the rat bladderoutflow obstruction model. (Pandita, R K, and Andersson K E. Effects ofintravesical administration of the K+ channel opener, Z.D6169, inconscious rats with and without bladder outflow obstruction. J Urol 162:943-948, 1999). Inflammatory models include injection of mustard oilinto the bladder.

To test the effectiveness of a TRPA1 inhibitor compound in treatingincontinence, varying concentrations of compound (e.g., low, medium, andhigh concentration) can be administered to rats following surgicalpartial bladder outlet obstruction (BOO). Efficacy of the varying dosesof TRPA1 inhibitory compound can be compared to controls administeredexcipients alone (sham control). Efficacy can further be compared torats administered a positive control, such as atropine. Atropine isexpected to decrease bladder over-activity following partial bladderoutlet obstruction in the BOO model. Note that when testing compounds inthe BOO model, compounds can be administered directly to the bladder orurethra (e.g., by catheter) or compounds can be administeredsystemically (e.g., orally, intravenously, intraperitoneally, etc).

As detailed above, TRPA1 inhibitors can be used to treat the symptoms ofpain associated with pancreatitis. The efficacy of TRPA1 inhibitors inpancreatitis pain management may be tested in one or more animal models.Inhibitors may be tested in general animal models of pain, for examplemodels of inflammatory pain or visceral pain. Alternatively oradditionally, TRPA1 inhibitors may be tested in animal models thatspecifically mimic pain accompanying pancreatitis or other pancreaticinjury.

Several rat models of pancreatic pain have recently been described (Lu,2003, Anesthesiology 98(3): 734-740; Winston et al., 2003, Journal ofPain 4(6): 329-337). Lu et al. induced pancreatitis by systemic deliveryof dibutylin dichloride in rats. Rats showed an increase in withdrawalevents after von Frey filament stimulation of the abdomen and decreasedwithdrawal latency after thermal stimulation during a period of 7 days.The pain state induced in these animals was also characterized byincreased levels of substance P in spinal cords (Lu, et al., 2003). Totest the efficacy of a TRPA1 inhibitor in this model, a TRPA1 inhibitorcan be administered following or concurrently with delivery of dibutylindichloride. Control animals can be administered a carrier or a knownpain reliever. Indicia of pain can be measured. Efficacy of a TRPA1inhibitor can be evaluated by comparing the indicia of pain observed inanimals receiving a TRPA1 inhibitor to that of animals that did notreceive a TRPA1 inhibitor. Additionally, efficacy of a TRPA1 inhibitorcan be compared to that of known pain medicaments.

The efficacy of von Frey filament testing as a means to measurenociceptive behavior was also shown by inducing pancreatitis by systemicL-arginine administration (Winston et al, 2003). The efficacy of a TRPA1inhibitor can similarly be tested following pancreatitis induced bysystemic L-arginine administration.

Lu et al. also described direct behavioral assays for pancreatic painusing acute noxious stimulation of the pancreas via an indwelling ductalcanula in awake and freely moving rats. These assays included cagecrossing, rearing, and hind limb extension in response tointrapancreatic bradykinin infusion. Intrathecal administration ofeither D-APV (NMDA receptor antagonist) or morphine alone partiallyreduced visceral pain behaviors in this model. Combinations of bothreduced pain behaviors to baseline. The efficacy of a TRPA1 inhibitorcan similarly be tested in this system.

Any of the foregoing animal models may be used to evaluate the efficacyof a TRPA1 inhibitor in treating pain associated with pancreatitis. Theefficacy can be compared to a no treatment or placebo control.Additionally or alternatively, efficacy can be evaluated in comparisonto one or more known pain relieving medicaments.

Optimizing the Treatment of Pain

TRPA1 inhibitors, according to the present invention, can be used in thetreatment of a variety of injuries, diseases, conditions, and disorders.One important therapeutic use for TRPA1 inhibitors is in the treatmentof pain. As illustrated by the extensive list of injuries, conditions,and diseases for which pain is a significant and sometimes debilitatingsymptom, improved methods and compositions for use in the treatment ofpain provide substantial benefits for an enormous range of patients.Such methods and compositions have the potential to improve the qualityof care and the quality of life for patients afflicted with a diverserange of injuries, diseases, and conditions. The present applicationcontemplates that a compound that inhibits TRPA1 can be used in thetreatment of any of the aforementioned injuries, conditions, ordiseases.

An important issue with the treatment of pain is how to manage painwhile reducing the side effects experienced with many analgesics. Forexample, although many opiates and other narcotics effectively diminishpain, patients are often unable to drive, work, or concentrate whiletaking these medications. Thus, while opiates such as morphine ordilaudin may be suitable for short term use or for use duringhospitalization, they are not optimal for long term use. Additionally,opiates and other narcotics are habit forming, and patients typicallydevelop a tolerance for these drugs. These characteristics of opioidsand other narcotics make them sub-optimal for pain management.

The present invention provides TRPA1 inhibitors for use in vitro and invivo. The present invention also provides compositions andpharmaceutical compositions comprising particular classes of compoundsthat inhibit TRPA1 activity. In certain embodiments, the subject TRPA1inhibitors are selective. In other words, in certain embodiments, thecompound inhibits TRPA1 activity preferentially over the activity ofother ion channels. In certain embodiments, the compound inhibits TRPA1activity preferentially over TRPV1, TRPV2, TRPV3, TRPV4, and/or TRPM8activity. In certain other embodiments, the compound is selected becauseit cross reacts with one or more other TRP channels involved with pain.For example, in certain embodiments, the compound inhibits the activityof TRPA1 and also inhibits the activity of one or more of TRPV1, TRPV2,TRPV3, TRPV4, and TRPM8.

Combination Therapy

Another aspect of the invention provides a conjoint therapy wherein oneor more other therapeutic agents are administered with the TRPA1modulators. Such conjoint treatment may be achieved by way of thesimultaneous, sequential, or separate dosing of the individualcomponents of the treatment.

In certain embodiments, a compound of the invention is conjointlyadministered with an analgesic. Suitable analgesics include, but are notlimited to, opioids, glucocorticosteroids, non-steroidalanti-inflammatories, naphthylalkanones, oxicams, para-aminophenolderivatives, propionic acids, propionic acid derivatives, salicylates,fenamates, fenamate derivatives, pyrozoles, and pyrozole derivatives.Examples of such analgesic compounds include, but are not limited to,codeine, hydrocodone, hydromorphone, levorpharnol, morphine, oxycodone,oxymorphone, butorphanol, dezocine, nalbuphine, pentazocine, etodolac,indomethacin, sulindac, tolmetin, nabumetone, piroxicam, acetaminophen,fenoprofen, flurbiprofen, ibuprofen, ketoprofen, naproxen, diclofenac,oxaprozin, aspirin, diflunisal, meclofenamic acid, mefanamic acid,prednisolone, and dexamethasone. Preferred analgesics are non-steroidalanti-inflammatories and opioids (preferably morphine).

In certain embodiments, a compound of the invention is conjointlyadministered with a non-steroidal anti-inflammatory. Suitablenon-steroidal anti-inflammatory compounds include, but are not limitedto, piroxicam, diclofenac, etodolac, indomethacin, ketoralac, oxaprozin,tolmetin, naproxen, flubiprofen, fenoprofen, ketoprofen, ibuprofen,mefenamic acid, sulindac, apazone, phenylbutazone, aspirin, celecoxiband rofecoxib.

In certain embodiments, a compound of the invention is conjointlyadministered with an antiviral agent. Suitable antiviral agents include,but are not limited to, amantadine, acyclovir, cidofovir, desciclovir,deoxyacyclovir, famciclovir, foscamet, ganciclovir, penciclovir,azidouridine, anasmycin, amantadine, bromovinyldeoxusidine,chlorovinyldeoxusidine, cytarbine, didanosine, deoxynojirimycin,dideoxycitidine, dideoxyinosine, dideoxynucleoside, edoxuidine,enviroxime, fiacitabine, foscamet, fialuridine, fluorothymidine,floxuridine, hypericin, interferon, interleukin, isethionate,nevirapine, pentamidine, ribavirin, rimantadine, stavirdine,sargramostin, suramin, trichosanthin, tribromothymidine,trichlorothymidine, vidarabine, zidoviridine, zalcitabine3-azido-3-deoxythymidine, 2′,3′-dideoxyadenosine (ddA),2′,3′-dideoxyguanosine (ddG), 2′,3′-dideoxycytidine (ddC),2′,3′-dideoxythymidine (ddT), 2′3′-dideoxy-dideoxythymidine (d4T),2′-deoxy-3′-thia-cytosine (3TC or lamivudime),2′,3′-dideoxy-2′-fluoroadenosine, 2′,3′-dideoxy-2′-fluoroinosine,2′,3′-dideoxy-2′-fluorothymidine, 2′,3′-dideoxy-2′-fluorocytosine,2′3′-dideoxy-2′,3′-didehydro-2′-fluorothymidine (Fd4T),2′3′-dideoxy-2′-beta-fluoroadenosine (F-ddA),2′3′-dideoxy-2′-beta-fluoro-inosine (F-ddI), and2′,3′-dideoxy-2′-beta-fluorocytosine (F-ddC), trisodiumphosphomonoformate, trifluorothymidine, 3′azido-3′thymidine (AZT),dideoxyinosine (ddI), and idoxuridine.

In certain embodiments, a compound of the invention is conjointlyadministered with an antibacterial agent. Suitable antibacterial agentsinclude, but are not limited to, amanfadine hydrochloride, amanfadinesulfate, amikacin, amikacin sulfate, amoglycosides, amoxicillin,ampicillin, amsamycins, bacitracin, beta-lactams, candicidin,capreomycin, carbenicillin, cephalexin, cephaloridine, cephalothin,cefazolin, cephapirin, cephradine, cephaloglycin, chilomphenicols,chlorhexidine, chloshexidine gluconate, chlorhexidine hydrochloride,chloroxine, chlorquiraldol, chlortetracycline, chlortetracyclinehydrochloride, ciprofloxacin, circulin, clindamycin, clindamycinhydrochloride, clotrimazole, cloxacillin, demeclocycline,diclosxacillin, diiodohydroxyquin, doxycycline, ethambutol, ethambutolhydrochloride, erythromycin, erythromycin estolate, erhmycin stearate,farnesol, floxacillin, gentamicin, gentamicin sulfate, gramicidin,giseofulvin, haloprogin, haloquinol, hexachlorophene, iminocylcline,iodochlorhydroxyquin, kanamycin, kanamycin sulfate, lincomycin,lineomycin, lineomycin hydrochloride, macrolides, meclocycline,methacycline, methacycline hydrochloride, methenine, methenaminehippurate, methenamine mandelate, methicillin, metonidazole, miconazole,miconazole hydrochloride, minocycline, minocycline hydrochloride,mupirocin, nafcillin, neomycin, neomycin sulfate, netimicin, netilmicinsulfate, nitrofurazone, norfloxacin, nystatin, octopirox, oleandomycin,orcephalosporins, oxacillin, oxyteacline, oxytetracycline hydrochloride,parachlorometa xylenol, paromomycin, paromomycin sulfate, penicillins,penicillin G, penicillin V, pentamidine, pentamidine hydrochloride,phenethicillin, polymyxins, quinolones, streptomycin sulfate,tetracycline, tobramycin, tolnaftate, triclosan, trifampin, rifamycin,rolitetracycline, spectinomycin, spiramycin, struptomycin, sulfonamide,tetracyclines, tetracycline, tobramycin, tobramycin sulfate,triclocarbon, triclosan, trimethoprim-sulfamethoxazole, tylosin,vancomycin, and yrothricin.

In certain embodiments, a compound of the invention is conjointlyadministered with a cough suppressant, decongestant, or expectorant.

Examples of retinoids that be administered with the subject TRPA1inhibitors, e.g., where the TRPA1 inhibitor can be used to reduce thepain and/or inflammatory effect of the retinoid, include, but are notlimited to, compounds such as retinoic acid (both cis and trans),retinol, adapalene, vitamin A and tazarotene. Retinoids are useful intreating acne, psoriasis, rosacea, wrinkles and skin cancers and cancerprecursors such as melanoma and actinic keratosis.

Similarly, the subject TRPA1 inhibitors can be used in conjunction withkeratolytic agents include benzoyl peroxide, alpha hydroxyacids, fruitacids, glycolic acid, salicylic acid, azelaic acid, trichloroaceticacid, lactic acid and piroctone.

The subject TRPA1 inhibitors can also be administered along withdepilatory agents (hair loss).

The subject TRPA1 inhibitors can be used with anti-acne agents,anti-eczema agents and anti-psoriatic agents. Compounds particularlyuseful in treating acne include azelaic acid (an aliphatic diacid withantiacne properties), anthralin (a diphenolic compound with antifungaland antipsoriatic properties), and masoprocol (nordihydroguairetic acid,a tetraphenolic compound with antioxidant properties, also useful in thetreatment of actinic keratosis) and analogs thereof (such asaustrobailignan 6, oxoaustrobailignan6,4′-O-methyl-7,7′-dioxoaustrobailignan 6, macelignan,demethyldihydroguaiaretic acid, 3,3′,4-trihydroxy-4′-methoxylignan,Saururenin, 4-hydroxy-3,3′,4′-trimethoxylignan, and isoanwulignan).Anti-eczema agents include pimecrolimus and tacrolimus. Anti-psoriaticactive agents suitable for use in the present invention includeretinoids (including isomers and derivatives of retinoic acid, as wellas other compounds that bind to the retinoic acid receptor, such asretinoic acid, acitretin, 13-cis-retinoic acid (isotretinoin),9-cis-retinoic acid, tocopheryl-retinoate (tocopherol ester of retinoicacid (trans- or cis-)), etretinate, motretinide,1-(13-cis-retinoyloxy)-2-propanone,1-(13-cis-retinoyloxy)-3-decanoyloxy-2-propanone,1,3-bis-(13-cis-retinoyloxy)-2-propanone,2-(13-cis-retinoyloxy)-acetophenone,13-cis-retinoyloxymethyl-2,2-dimethyl propanoate,2-(13-cis-retinoyloxy)-n-methyl-acetamide,1-(13-cis-retinoyloxy)-3-hydroxy-2-propanone,1-(13-cis-retinoyloxy)-2,3-dioleoylpropanone, succinimdyl13-cis-retinoate, adapalene, and tazarotene), salicylic acid(monoammonium salt), anthralin, 6-azauridine, vitamin D derivatives(including but not limited to Rocaltrol (Roche Laboratories), EB 1089(24α,26α,27α-trihomo-22,24-diene-1α,25-(OH)₂-D₃), KH 1060(20-epi-22-oxa-24α,26α,27α-trihomo-1α,25-(OH)₂-D₃), MC 1288, GS 1558, CB1093, 1,25-(OH)₂-16-ene-D₃, 1,25-(OH)₂-16-ene-23-yne-D₃, and25-(OH)2-16-ene-23-yne-D₃, 22-oxacalcitriol; 1α-(OH)D₅ (University ofIllinois), ZK 161422 and ZK 157202 (Institute of MedicalChemistry-Schering AG), alfacalcidol, calcifediol, calcipotriol(calcipotriene), maxacalcitriol, colecalciferol, doxercalciferol,ergocalciferol, falecalcitriol, lexacalcitol, maxacalcitol,paricalcitol, secalciferol, seocalcitol, tacalcitol, calcipotriene,calcitriol, and other analogs as disclosed in U.S. Pat. No. 5,994,332),pyrogallol, and tacalcitol.

The subject TRPA1 inhibitors can also be administered with vitamins andderivatives thereof including Vitamin A, ascorbic acid (Vitamin C),alpha-tocopherol (Vitamin E), 7-dehydrocholesterol (Vitamin D), VitaminK, alpha-lipoic acid, lipid soluble anti-oxidants, and the like.

The subject TRPA1 inhibitors can also be used with skin protectants,such allantoin and esculin.

In certain embodiments, two or more compounds of the invention areconjointly administered. When two or more compounds of the invention areconjointly administered, the two or more compounds may have a similarselectivity profile and functional activity, or the two or morecompounds may have a different selectivity profile and functionalactivity. By way of example, the two or more compounds may both beapproximately 10, 100, or 1000 fold selective for antagonizing afunction of TRPA1 over TRPV1, TRPV5, and TRPV6 (e.g., the two or morecompounds have a similar selectivity profile), and further may inhibit afunction of TRPA1 with a similar IC50 (e.g., a similar functionalactivity). Alternatively, the one of the two or more compounds mayselectively inhibit TRPA1 while the other of the two or more compoundsinhibits both TRPA1 and TRPV1 (e.g., the two or more compounds havediffering selectivity profiles). Administration of combinations of twoor more compounds of the invention having similar or differingproperties are contemplated.

In certain embodiments, a compound of the invention is conjointlyadministered with one or more additional compounds that antagonize thefunction of a different channel. By way of example, a compound of theinvention may be conjointly administered with one or more compounds thatantagonize TRPV1, TRPM8, and/or TRPV3. The compound(s) that antagonizeTRPV1, TPRM8, or TRPV3 may be selective for TRPV1, TRPM8 or TRPV3 (e.g.,inhibit TRPV1 or TRPV3 10, 100, or 1000 fold more strongly than TRPA1).Alternatively, the compound(s) that antagonize TRPV1 or TRPV3 may crossreact with other TRP channels.

In certain other embodiments, a compound of the invention is conjointlyadministered with one or more additional agents or therapeutic regimensappropriate for the particular injury, disease, condition, or disorderbeing treated.

Pharmaceutical Compositions

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The compounds according to theinvention may be formulated for administration in any convenient way foruse in human or veterinary medicine. In certain embodiments, thecompound included in the pharmaceutical preparation may be activeitself, or may be a prodrug, e.g., capable of being converted to anactive compound in a physiological setting.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms such as described below orby other conventional methods known to those of skill in the art.

Thus, another aspect of the present invention provides pharmaceuticallyacceptable compositions comprising a therapeutically effective amount ofone or more of the compounds described above, formulated together withone or more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail below, the pharmaceutical compositionsof the present invention may be specially formulated for administrationin solid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular or intravenous injection as, forexample, a sterile solution or suspension; (3) topical application, forexample, as a cream, ointment or spray applied to the skin; (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; or (5) for inhalation. However, in certain embodiments the subjectcompounds may be simply dissolved or suspended in sterile water. Incertain embodiments, the pharmaceutical preparation is non-pyrogenic,i.e., does not elevate the body temperature of a patient.

The phrase “therapeutically effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect by inhibiting TRPA1 function in at least asub-population of cells in an animal and thereby blocking the biologicalconsequences of that function in the treated cells, at a reasonablebenefit/risk ratio applicable to any medical treatment.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject antagonistsfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about1 percent to about ninety-nine percent of active ingredient, preferablyfrom about 5 percent to about 70 percent, most preferably from about 10percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

It is known that sterols, such as cholesterol, will form complexes withcyclodextrins. Thus, in preferred embodiments, where the inhibitor is asteroidal alkaloid, it may be formulated with cyclodextrins, such as α-,β- and γ-cyclodextrin, dimethyl-β-cyclodextrin and2-hydroxypropyl-β-cyclodextrin.

Formulations of the pharmaceutical compositions of the invention forrectal, vaginal, or urethral administration may be presented as asuppository, which may be prepared by mixing one or more compounds ofthe invention with one or more suitable nonirritating excipients orcarriers comprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Alternatively or additionally, compositions can be formulated fordelivery via a catheter, stent, wire, or other intraluminal device.Delivery via such devices may be especially useful for delivery to thebladder, urethra, ureter, rectum, or intestine.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that may berequired.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”,W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feedsand Feeding” 0 and B books, Corvallis, Ore., U.S.A., 1977).

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinaceous biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a compound at a particular targetsite.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient will range from about 0.0001 to about 100 mg perkilogram of body weight per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable and/or sterile carriers andcan also be administered in conjunction with other antimicrobial agentssuch as penicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticeffects of the first administered one are still detectable when thesubsequent therapy is administered.

The present invention contemplates formulation of the subject compoundsin any of the aforementioned pharmaceutical compositions andpreparations. Furthermore, the present invention contemplatesadministration via any of the foregoing routes of administration. One ofskill in the art can select the appropriate formulation and route ofadministration based on the condition being treated and the overallhealth, age, and size of the patient being treated.

EXAMPLES Example 1 High Throughput Screening Assay

The assay depended on detection of the rise in intracellular Ca²⁺concentration ([Ca²+]_(i)) following channel activation in cellsinducibly expressing the TRPA1 channel. Ca²⁺ rise was quantified withthe use of fluorescent Ca²⁺ indicators that were loaded into cells andthereafter indicated the [Ca²⁺]_(i). Ca²⁺ influx followed activation ofthe TRPA1 channel. Compounds inhibiting the [Ca²⁺]_(i) rise wereconsidered hits for further investigation.

The commercially available HEK293/TREx line (Invitrogen) was stablytransfected with a TRPA1 construct (specifically a construct encoding aTRPA1 protein with an amino acid sequence depicted in SEQ ID NO: 1) andscreened by conventional calcium imaging to find clones with TRPA1expression following stimulation with 1 μg/ml tetracycline. These cellswere maintained in the growth medium recommended by the manufacturersupplemented with 100 μg/ml hygromycin to promote retention of the TRPA1construct. After growing to near confluency, cells were plated at adensity of ˜25,000 cells/well in 384 well CellBind plates (Corning) inthe presence of 1 μg/ml tetracycline, and allowed to grow for 20-30 hrs.A nearly confluent monolayer resulted. Cells were then loaded with Ca²⁺dye: Fura-2/AM or Fluo4/AM was added to the wells to a finalconcentration of 2 μM or 1 μM, respectively, and incubated for 80 min or60 min, respectively, at room temperature. Supernatant was then removedfrom the cells by inverting plates with a sharp flick, and 40 μl Hank'sBalanced Salt Solution (HBSS; 0.185 g/l D-glucose, 0.9767 g/l MgSO₄(anhydrous), 0.4 g/l KCl, 0.06 g/l KH₂PO₄ (anhydrous), 0.35 g/l NaHCO₃,8.0 g/l NaCl, and 0.04788 g/l Na₂HPO₄ (anhydrous); pH 7.4) was thenadded to each well. Following ˜1 hour for recovery from loading, cellswere assayed using the Hamamatsu FDSS 6000 system, which permittedillumination alternately at 340 nM and 380 nM for Fura-2 experiments, orat 485 nM for Fluo4 experiments. Frames were acquired at a rate of 0.2Hz. During the assay, the plates were continuously vortexed, withpipette mixing of wells following addition of each reagent. For thescreening assay, 13 μl of a diluted stock (at 50 μM) was added to eachwell for 2 minutes following the collection of a short (4 frame)baseline. 13 μl 37.5 μM AITC (allylisothiocyanate) was then added toeach well, achieving a final concentration of 10 μM each compound and7.5 μM AITC. Data was collected for ˜3 minutes following addition ofAITC, where the fluorescent intensity (for Fluo4) and the F340/F380ratio (for Fura-2) were proportional to the [Ca²⁺]_(i). Negativecontrols consisted of HEK293/TREx TRPA1 cells exposed to AITC, but nocompound. Positive control cells were usually HEK293/TREx (“parental”)cells exposed to AITC but no compound, but sometimes normal HEK/293 TRExTRPA1 cells were also used, but not exposed to AITC or compound. Thesecontrols defined a screening window, and “hits” were defined as thosecompounds inhibiting the fluorescence response by at least 40%. IC₅₀values were determined for compounds defined as “hits.” The Fluo4cell-based fluorescence assay was used to determine the intracellularCa²⁺ concentration in the presence of varying drug concentration.Concentrations tested were 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM,and 0.625 μM. Compounds were tested in triplicate at all concentrations.Standard software was used to fit IC₅₀ curves.

Additionally or alternatively, efficacy can be represented as %inhibition in the presence (of a given concentration of compound) versusthe absence of compound or in comparison to a control compound. Forexample, efficacy can be represented as % inhibition of ion flux in thepresence versus the absence of compound.

Example 2 Patch Clamp Experiments

Patch clamp experiments permit the detection of currents through theTRPA1 channel in the cell line described above. To permit recording ofcurrent at a stable level and prevent the “rundown” observed by otherlabs, it is necessary to use the perforated patch technique, whichprevents dialysis of the cytoplasm with the pipette solution. In normalwhole-cell patch clamp recordings, a glass electrode is brought intocontact with a single cell and a high-resistance (gigaohm) seal isestablished with the cell membrane. The membrane is then ruptured toachieve the whole-cell configuration, permitting control of the voltageof the cell membrane and measurement of currents flowing across themembrane using the amplifier attached to the electrode and resulting inthe replacement of cytoplasm with the pipette solution. In contrast, inthe perforated patch mode, an antibiotic, amphotericin, is present inthe pipette solution and diffuses into contact with the cell after theseal is achieved, over the course of several minutes. The amphotericinforms ion-permeable pores in the membrane under the pipette, permittingpassage of some ions but maintaining most native cytosolic components. Aperfusion system permits control of the extracellular solution,including the addition of blockers and activators of the current. Thecurrent can be activated by addition of 5 μM AITC to the solution.

TRPA1 cells were induced 20-48 hours, removed from growth plates, andreplated at low density (to attain good single-cell physical separation)on glass coverslips for measurement. In some cases, cells were grown inlow density overnight on glass coverslips. Patch clamp recordings weremade in the whole-cell mode with a holding potential of −40 mV. Every 5seconds, a voltage ramp was applied from −120 to +100 mV, 400 ms induration. Currents elicited were quantified at −80 mV and +80 mV. Theinternal solution consisted of 140 mM cesium aspartate, 10 mM EGTA, 2.2mM CaCl₂, 2.08 mM MgCl₂ and 10 mM HEPES, pH 7.2, with 50 nM calculatedfree Ca²⁺ and 60 mg/ml amphotericin added immediately prior toexperiments. The external solution consisted of 150 mM NaCl, 4.5 mM KCl,3 mM MgCl₂, 10 mM HEPES, 10 mM glutamine, 1 mM EGTA, pH 7.4. Uponaddition of AITC, TRPA1 current was induced only in TRPA1-expressingcells and not in parental HEK293 TREx cells. Removal of the AITCstimulus causes most of the current to go away. Potential blockers weretested for ability to block both inward and outward currents in thecontinued presence of AITC.

IC₅₀ of compounds was estimated by testing each compound at 5 μM and 500nM. When 5 μM compound showed no block, IC₅₀ was estimated as >10 μM.When 5 μM compound showed 50% or less block, a rough estimate of IC₅₀ inthe range of 5-10 μM could be made. IC₅₀ for compounds between 500 nMand 5 μM was similarly estimated. Compounds blocking 50% or more at 500nM are retested at multiple concentrations, and the % block at each isfitted by standard equations to determine IC₅₀ accurately using a 5-6point concentration/response experiment. Except where indicated, theIC₅₀ values presented in Tables 1, 2, 3, and 4 were obtained from patchclamp experiments.

Example 3 Other Screening Assays

Although the exemplary TRPA1 inhibitors provided herein were identifiedusing the assays described in Examples 1 and 2, other cell-based assayscan be used to identify and/or characterize TRPA1 inhibitors. One suchassay is described in U.S. application Ser. No. 11/078,188, filed Mar.11, 2005, the contents of which are hereby incorporated by reference intheir entirety. TRPA1 protein can be expressed in the prokaryotic cellsystem described in application Ser. No. 11/078,188, and this system canbe used to screen for compounds that modulate an activity of the TRPA1protein. Alternatively, an ion channel other than TRPA1 can be expressedin the prokaryotic cell system, and the system can be used to evaluatethe activity profile of an identified TRPA1 inhibitors with respect toother ion channels.

Any assays performed to identify and/or characterize compounds thatinhibit an activity of TRPA1 can be performed in a high-throughputfashion, or can be performed on a smaller scale examining individualcompounds or small numbers of compounds. Additionally, any of theseassays can be performed (i) as a primary assay to identify compoundsthat inhibit a function of TRPA1; (ii) as a secondary assay to assessthe specificity of a compound with respect to its activity against otherion channels; (iii) as an assay used in a medicinal chemistry program tooptimize subject compounds.

Example 4 Testing of TRPA1 Antagonists in a Thermal Injury Model of Pain

The thermal injury model can be used to evaluate the effectiveness of anexemplary TRPA1 inhibitor in the treatment of nociceptive pain using thefollowing protocol. Male Holtzman rats (approximately 300 grams) aretested on thermal escape using a Hargreaves type apparatus. Under lightanesthesia, a thermal injury (52° C. for 45 seconds) is applied to oneheel. The animals are tested for thermal escape latency of the injuredand uninjured paw before and at 30, 60, 80, and 120 minutes afterinjury. Drug (a TRPA1 inhibitor) or vehicle (0.5% methylcellulose) isadministered after the baseline measurement and approximately 15-20minutes prior to the thermal injury. In addition to the escape latencymeasurement, behavioral observations are made throughout the experiment.

Example 5 Testing of TRPA1 Antagonists in the Chung Model of NeuropathicPain

Briefly, male Sprague Dawley rats (approximately 175 grams) are preparedwith ligation of the L4/5 nerve roots. After 5-8 days, the animals aretested for tactile allodynia using Von Frey hairs. Thresholds areassessed with the “up-down” method. Drug or vehicle is administered andthe animals tested periodically over the next four hours.

Example 6 Synthetic Methods

General Procedure A for the Preparation of Amides by Coupling Using EDCI

To a mixture of theophylline-7-acetic acid (2 mmol), DMAP (2 mmol),substituted phenethylamine (2 mmol) and DIPEA (4 mmol) in DMF (20 mL) isadded EDCI (2 mmol). The reaction mixture is heated to 40° C. andstirred over night. The solution is concentrated in vacuo and theresidue is dissolved in EtOAc (100 mL), washed with H₂O, citric acid(10%), NaHCO₃ (sat.) and brine, dried over Na₂SO₄ and concentrated invacuo. The crude product is purified by flash chromatography on silicagel eluting with MeOH/EtOAc (1˜8%).

General Procedure B for the Preparation of Amides Via Acid Chloride

A suspension of theophylline-7-acetic acid (2 mmol) in CHCl₃ (15 mL) andMeCN (15 mL) is cooled in an ice-water bath. Oxalyl chloride (2.2 mmol)is then added dropwise. Catalytic DMF (˜25 μL) is then added. Themixture is stirred at room temperature over night. The solution is thencooled in an ice-water bath, and DMAP (2.5 mmol) is added in oneportion. The substituted phenethylamine is added dropwise and thereaction mixture is stirred at room temperature over night. Afterdiluting with CHCl₃ (50 mL), the mixture is washed with H₂O, citric acid(10% in H₂O), NaHCO₃ (sat.), dried over Na₂SO₄ and concentrated invacuo. The crude product is purified by flash chromatography on silicagel eluting with MeOH/EtOAc (1˜8%).

Dihydropyrimidine-dione 2 can be prepared by reacting 1-propylurea (1)and ethyl 2-cyanoacetate, which can be subsequently treated withbromine, ethyl 2-aminoacetate, and triethoxymethane to yield compound 6,ethyl 2-(2,6-dioxo-1-propyl-2,3-dihydro-1H-purin-7(6H)-yl)acetate. Theobtained dihydropurine 6 can be transformed to compound 10 throughmethylation, hydrolysis, and a coupling reaction under CDI.

Dihydropyrimidine-dione 2 can be prepared by reacting 1-propylurea (1)and ethyl 2-cyanoacetate, which can be subsequently treated withbromine, ethyl 2-aminoacetate, and triethoxymethane to yield compound 6,ethyl 2-(1-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetate. Theobtained dihydropurine 6 can be converted to compound 10 throughalkylation reaction, hydrolysis, and a coupling reaction catalyzed byCDI.

N,N-Dimethylethane-1,2-diamine can be converted to urea 2, which canthen react with ethyl 2-cyanoacetate to give dihydropyrimidine-dione 3.Compound 3 can be subsequently treated with bromine, ethyl2-aminoacetate, and triethoxymethane to yield compound 7, ethyl2-(1-(2-(dimethylamino)ethyl)-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetate.The obtained dihydropurine 7 can be transformed to compound 11 throughalkylation reaction, hydrolysis, and a coupling reaction catalyzed byCDI.

Compound 2 can be prepared by coupling dihydropurine 1 with2-p-tolylethanol.

Esterification of dihydropurine 1, followed by reduction with LAH, Swernoxidation and coupling reaction can yield compound 5, which subsequentlycan be converted to compound 6, compound 7, and compound 8 throughmethylation, acylation, or sulphonylation.

Dihydropurine 1 can be coupled with 2-(4-methylpiperazin-1-yl)ethanamineby CDI to give compound 2.

2-(4-Phenylpiperazin-1-yl)ethanamine 4 can be prepared by reacting1-phenylpiperazine with 2-chloroacetamide, followed by a reductionreaction with LAH. Amine 4 then can be coupled with dihydropurine 5 toyield compound 6.

2-(1-Benzyl-1H-imidazol-2-yl)ethanamine 4 can be prepared by protectionof imidazole, followed by alkylation and a deprotection reaction withTFA. Amine 4 then can be coupled with dihydropurine 5 to afford compound6.

Treatment of imidazole 1 with n-BuLi, followed by an alkylationreaction, and a deprotection reaction with TFA affords2-(1-methyl-1H-imidazol-2-yl)ethanamine. Amine 3 can be coupled withdihydropurine 4 to give compound 5.

2-(Thiazol-2-yl)ethanamine 3 can be prepared by treatment of thiazole 1with n-BuLi, followed by addition of Boc-protected 2-bromoethanamine anda deprotection reaction with TFA. The obtained amine 3 then can becoupled with carboxylic acid 4 to afford compound 5.

Treatment of oxazole 1 with n-BuLi, followed by an alkylation reaction,and a deprotection reaction with TFA affords 2-(oxazol-2-yl)ethanamine3. Amine 3 can be coupled with dihydropurine 4 to give compound 6.

Compound 10 (Scheme 12) can be prepared according to similar reactionprocedures shown in Scheme 1.

Treatment of Protected indoline 2 with LDA and dibromopropane givescompound 3, which subsequently can react with purine-dione 4, followedby hydrolysis reaction to yield compound 6.

p-Tolylmethanol (1) can be converted to 1-(bromomethyl)-4-methylbenzene2, which can be treated with Mg and allyl bromide to give compound 3.Treatment of alkene 3 with N₂CH₂CO₂Et, followed by a reduction reactionaffords (2-(4-methylphenethyl)cyclopropyl)methanol (5).Cyclopropylmethanol 5 can react with MsCl, and the resulting compound 6can be coupled with purine-dione 7 to afford compound 8.

2-(5-Methylpyridin-2-yl)ethanamine 4 can be prepared by converting2-chloro-5-methylpyridine (1) to 2-bromo-5-methylpyridine, followed byreacting with Boc-protected 2-bromoethanamine and removal of theprotecting group with TFA. The obtained amine 4 then can be coupled withcarboxylic acid 5 to afford compound 6.

LAH can reduce ethyl 6-methylnicotinate to give alcohol 2, which can beoxidized and subsequently treated with MeNO₂ to yield compound 4.Compound 4 can be reduced to amine 4, which can be coupled withcarboxylic acid 6 to give compound 7.

Example 7 Synthesis ofN-(4-(4-(diethylamino)phenyl)thiazol-2-yl)-2-(1,3-dimethyl-2,6-dioxo-3,4,5,6-tetrahydro-1H-purin-7(2H)-yl)acetamide

To a solution of 4′-diethylamionoacetophenone (20.80 g, 0.109 mol) in 45mL HBr (48% in water), a solution of bromine (5.50 mL, 0.109 mol) in 35mL HBr was added slowly via addition funnel over 20 min. Reactionmixture was stirred at room temperature overnight, diluted with 300 mLwater and poured onto NaHCO₃/ice mixture. The mixture was extracted withCHCl₃ (2×400 mL), and the combined organic phase was washed with brine,and then dried over Na₂SO₄. After evaporation, the green oil wasdissolved in 120 mL EtOH followed by addition of thiourea (8.30 g, 0.109mol) and the solution was refluxed for 2 hrs. After evaporation of about50 mL EtOH on rotovap, lot of solid precipitated in the flask. Themixture was filtered, washed with EtOH (100 mL), and then dried undervacuum to get brownish solid 20.86 g (77%). Ref: J. Org. Chem. 2003, 68,839-853.

In an oven-dried round-bottom flask, theophylline-7-acetic acid (3.18 g,13.3 mmol) and triethylamine (2.5 mL, 18.2 mmol) was dissolved in 60 mLDMF, and then 4-(diethylamino-phenyl)-thiazol-2-ylamine (3.00 g, 12.1mmol) was added. After the amine totally dissolved, the solution wascooled in an ice-water bath for 20 min, and then HATU was added in oneportion. The reaction mixture was warmed up to room temperaturegradually and stirred at this temperature for 90 min. Mass and TLCshowed consumption of amine. The solution was poured into 500 mL brineat 0° C., and the cloudy suspension was stirred for 30 min at thistemperature. The suspension was filtered and washed with water andether. Solid was dried in oven (50° C.) for 2 h, the off-white solid wasthen suspended in 500 mL EtOAc/10% MeOH and refluxed for 2 hrs. Hotfiltration was performed, and the solid was washed with ether and driedon vacuum to give white solid 4.00 g (71%). mp: 305-307° C. R_(f)=0.31(EtOAc).

Incorporation by Reference

All publications and patents mentioned herein, are hereby incorporatedby reference in their entirety as if each individual publication orpatent was specifically and individually indicated to be incorporated byreference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A compound of formula (VIII), or a pharmaceutically acceptable saltthereof:

wherein, each of R¹ and R² is independently, C₁-C₆ alkyl; L is C(O)NR⁶;R^(3a) is an optionally substituted nitrogen-containing heteroaryl,R^(3b) is aryl or heteroaryl, each of which is optionally substitutedwith 1-4 R⁷; R⁶ is H; each R⁷ is independently heterocyclyl, which isoptionally substituted with 1-3 R⁸; each R⁸ is independently C₁-C₆alkyl; R⁹ is H; and m is 1, provided that the compound is not a compounddepicted in Table
 2. 2. The compound of claim 1, wherein R^(3a) ispyridyl.
 3. The compound of claim 1, wherein R^(3b) is phenyl.
 4. Thecompound of claim 1, wherein R¹ and R² are both methyl.
 5. The compoundof claim 1, wherein R3B is substituted with 1-4 R7.